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Immunization with Apoptotic Phagocytes Containing Histoplasma Activates 1

Functional CD8+ T Cells to Protect Against Histoplasmosis 2

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Running title: Effective Immunization Protects Against Histoplasmosis 4

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Shih-Hung Hsieh,1 Jr-Shiuan Lin,1† Juin-Hua Huang,1 Shang-Yang Wu1,Ching-Liang 6

Chu, 1, 2, 3 John T. Kung,4 and Betty A. Wu-Hsieh1* 7

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Graduate Institute of Immunology, National Taiwan University College of Medicine, 9

Taipei, Taiwan1; Immunology Research Center, National Health and Research Institutes, 10

Miaoli, Taiwan2; Yeastern Biotech Co., Taipei County, Taiwan 3; Institute of Molecular 11

Biology, Academia Sinica, Nankang, Taipei, Taiwan4 12

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* Corresponding author. Mailing address: Graduate Institute of Immunology, National 14

Taiwan University College of Medicine, No. 1, Ren-Ai Road, Section 1, Taipei, Taiwan. 15

Phone: 886-2-321-7510. Fax: 886-2-2321-7921. E-mail: [email protected]. 16

† Present address: Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY12983, 17

USA. 18

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.05350-11 IAI Accepts, published online ahead of print on 12 September 2011

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ABSTRACT 19

We have previously revealed the protective role of CD8+ T cells in host defense against 20

Histoplasma capsulatum in animals with CD4+ T cell deficiency and demonstrated that 21

sensitized CD8+ T cells are restimulated in vitro by dendritic cells that have ingested 22

apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization 23

with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated 24

functional CD8+ T cells which contributed equally to CD4+ T cells in protection against 25

Histoplasma challenge. Inhibition of macrophage apoptosis due to iNOS deficiency or by 26

caspase inhibitor treatment dampened the CD8+ T cell but not CD4+ T cell response to 27

pulmonary Histoplasma infection. In mice subcutaneously immunized with viable 28

Histoplasma yeasts where CD8+ T cells are protective against Histoplasma challenge, 29

there was heavy granulocyte and macrophage infiltration and the infiltrating cells became 30

apoptotic. In mice subcutaneously immunized with CFSE-labeled apoptotic macrophages 31

containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to 32

localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic 33

cells in immunized CD11c-DTR mice significantly reduced CD8+ T cell activation. Taken 34

together, our results revealed that phagocyte apoptosis in Histoplasma-infected host is 35

associated with CD8+ T cell activation and that immunization with apoptotic phagocytes 36


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containing heat-killed Histoplasma efficiently evokes a protective CD8+ T cell response. 37

These results suggest that employing apoptotic phagocytes as antigen donor cells is a 38

viable proposition to develop efficacious vaccines to elicit strong CD8+ T cell as well as 39

CD4+ T cell responses against Histoplasma infection. 40

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INTRODUCTION 42

Histoplasma capsulatum is an opportunistic fungal pathogen that threatens the life of 43

immune compromised individuals, especially those infected with HIV (8). Upon entry into the 44

respiratory system, the fungus transforms into yeast cell and is phagocytosed by the 45

macrophage. The yeast cells of Histoplasma reside and replicate primarily in the phagosome of 46

macrophages (16). IFN-γ produced by CD4+ and CD8+ T cells can activate macrophages to 47

produce reactive oxygen species and nitric oxide for fungal clearance (3, 14, 16, 31). Depletion 48

study has established the vital role of CD4+ T cells in clearing Histoplasma in intranasal 49

infection of the wild-type mice (3). Depletion of CD8+ T cells or deficiency in MHC class I, on 50

the other hand, has little effect on fungal clearance (3, 5). However, the protective role of CD8+ 51

T cells stands out in infection of mice with MHC class II deficiency (14), demonstrating that 52

CD8+ T cells can be protective against histoplasmosis in the absence of functional CD4+ T 53

cells. In HIV-infected individuals whose CD4+ T cell responses gradually decline, the CD8+ T 54

cells become the major effectors to defend against opportunistic infections. Thus, developing 55

vaccines that aim to induce functional CD8+ T cell immune responses will be valuable. 56

Vaccines designed for non-viral pathogens often focus on eliciting CD4+ T cell and B cell 57

immune responses (15, 24). CD8+ T cell response receives relatively little attention. Recently, 58

increasing evidences demonstrate that exogenous or non-viral antigens can be presented on 59


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MHC class I molecules to prime CD8+ T cell responses, processes referred to as 60

“cross-presentation” and “cross-priming” (12). Exogenous antigens coupled with heat shock 61

protein (25), exosomes (29), immune complexes (20) or latex beads (22) all can be 62

cross-presented to prime CD8+ T cells. Immunizing mice with antigen-containing dead cells or 63

with dead cell-pulsed dendritic cells is a well-recognized strategy in the development of cancer 64

vaccines to elicit strong CD8+ T cell responses (6, 9). In the studies of infectious diseases, 65

Albert et al. were the first to report that apoptotic monocytes deliver influenza antigens to 66

dendritic cells and trigger CD8+ T cell immune responses (1). Non-viral intracellular pathogens 67

such as Salmonella and Mycobacterium induce macrophage apoptosis and the apoptotic cell 68

blebs shuttle the bacterial antigens to uninfected bystander antigen-presenting cells to 69

cross-prime CD8+ T cells (21, 33). We previously showed that sensitized CD8+ T cells are 70

restimulated in vitro by dendritic cells that acquired Histoplasma antigens through 71

phagocytosis of heat-killed Histoplasma-containing apoptotic macrophages (14). In addition, 72

Winau et al. showed that subcutaneously immunizing mice with apoptotic vesicles prepared 73

from BCG-OVA-infected macrophages not only induces adoptively transferred OT-1 CD8+ T 74

cell division and IFN-γ production in vivo but also protects mice from tuberculosis (28). These 75

studies together raise the possibility that the similar strategy aiming to cross-prime strong 76

CD8+ T cell responses could be applied to develop fungal vaccines. 77


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Here we showed that immunization with apoptotic phagocytes (macrophages or neutrophils) 78

containing heat-killed Histoplasma efficiently activated functional CD8+ and CD4+ T cells. 79

Inhibiting apoptosis during early phase of infection weakened the CD8+ T cell but not CD4+ T 80

cell response to pulmonary Histoplasma infection. We also demonstrated that in mice 81

subcutaneously immunized with viable Histoplasma yeasts where CD8+ T cells are protective, 82

there was heavy granulocyte and macrophage infiltration and the infiltrating cells became 83

apoptotic. While the CFSE-labeled macrophage material localized within dendritic cells in the 84

draining lymph node after immunization with CFSE-labeled [Apoptotic pMac (HK Hc)], 85

depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8+ T cell 86

activation. Our results revealed that phagocyte apoptosis during early phase in 87

Histoplasma-infected host is associated with CD8+ T cell activation and that immunization 88

with [Apoptotic pMac (HK Hc)] or [Apoptotic pNeu (HK Hc)] efficiently evokes protective 89

CD8+ T cell response. Thus, it is a viable option to exploit apoptotic phagocytes as antigen 90

donor cells in the development of vaccines that elicit strong protective CD8+ T cell as well as 91

CD4+ T cell response against Histoplasma. 92

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MATERIALS AND METHODS 94

Mice. Wild-type, iNOS-/- and β2m-/- mice were originally obtained from the Jackson 95

Laboratory (Bar Harbor, Maine, USA) and bred at Laboratory Animal Center, National Taiwan 96

University College of Medicine. CD11c-DTR transgenic mice (10) were provided by Dr. A. 97

DeFranco (UCSF, San Francisco, CA). All mice used were of C57BL/6 background and were 98

housed in sterilized cages fit with filter cage tops and fed with sterilized food and water. Mice 99

at 8–10 wk of age were used in all the experiments in this study. This study was carried out in 100

strict accordance with the recommendations in the Guidebook for the Care and Use of 101

Laboratory Animals, The Third Edition, 2007, published by The Chinese-Taipei Society of 102

Laboratory Animal Sciences. The protocol was approved by the Committee on the Ethics of 103

Animal Experiments of the National Taiwan University College of Medicine (Permit Number: 104

20040318) 105

Antibodies and medium. APC-rat-anti-mouse CD8 (clone 53-6.7), FITC-rat-anti-mouse 106

CD4 (clone RM4-5), PE-hamster-anti-mouse CD3 (clone 145-2C11), FITC-rat-anti-mouse 107

B220 (clone RA3-6B2), PE-rat-anti-mouse F4/80 (clone BM8), PE-rat-anti-mouse Gr-1 (clone 108

RB6-8C5) and PE-rat IgG1 isotype (for immunofluorescence staining) antibodies were 109

purchased from eBioscience (San Diego, CA). FITC- and PE-hamster-anti-mouse CD11c 110

(clone HL3) were from BD Pharmingen (San Diego, CA). Purified hamster-anti-CD3 (clone 111


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145-2C11), purified hamster-anti-mouse CD28 (clone 37.51) and PE-rat-anti-mouse IFN-γ 112

(clone XMG1.2) antibodies were from BioLegend (San Diego, CA). PE-mouse-anti-human 113

granzyme B (clone MHGB04) antibody was from Caltag Laboratories (Burlingame, CA). 114

RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) was supplemented with 10% 115

heat-inactivated fetal calf serum (Biological Industries, Israel), 2 mM L-glutamine, 0.1 mM 116

nonessential amino acid, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 117

5×10-5 M 2-mercaptoethanol and 25 mM HEPES buffer. The supplemented medium was used 118

as RPMI 1640 complete medium. All of the supplements except serum were obtained from 119

Gibco-BRL. 120

Fungus and infection. Histoplasma capsulatum strain 505 yeast cells were cultured at 121

37°C on brain-heart infusion agar (DIFCO, Sparks, MD) supplemented with 1 mg/ml cysteine 122

and 20 mg/ml glucose. Fresh yeast cell suspensions were prepared in RPMI 1640 medium for 123

infection. For intratracheal infection, a small incision was made on the skin of mice under 124

anesthesia to expose the trachea. Two hundred thousand (2×105) of Histoplasma yeasts in 40 μl 125

RPMI 1640 medium were injected into the trachea through a 29-gauge insulin syringe. The 126

skin was sutured and the mice were kept under a warming lamp until come to. For 127

subcutaneous infection, two million (2×106) Histoplasma yeasts were suspended in 100 μl 128

RPMI 1640 medium. Mice were anesthetized and each side of the base of the tail was 129


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inoculated with 50 μl of Histoplasma suspension through a 29-gauge insulin syringe. 130

Heat-killed Histoplasma yeast cells were prepared by heating fresh prepared yeasts at 65°C for 131

120 min. 132

Intracellular IFN-γ and granzyme B staining. Single cell suspensions of the inguinal or 133

mediastinal lymph nodes were prepared. One million lymph node cells in RPMI 1640 134

complete medium were cultured with 1×105 heat-killed Histoplasma yeast cells and 1×106 135

irradiated (1000 Rads) spleen cells from naive mice for 24 h. At 6 h before harvest, monensin 136

(2 µM; Sigma-Aldrich, St. Louis, MO) was added to the culture to block intracellular protein 137

transport. Cells were collected and stained for surface CD4 and CD8 and intracellular IFN-γ as 138

previously described (13). For granzyme B staining, the cells were first incubated with 2.4G2 139

Ab (BioLegend) directed against mouse Fcγ II/III receptors before staining with 140

PE-anti-granzyme B mAbs as previously described (14). PE-mouse IgG1 was used as isotype 141

control. 142

Inguinal lymph node cells harvested from CD11c-DTR transgenic mice were stimulated 143

with plate-bound anti-mouse CD3 (0.5 µg/ml) and anti-mouse CD28 (0.5 µg/ml) mAbs for 5 h 144

in the presence of monesin. Cells were collected and stained for surface CD4 and CD8 and 145

intracellular IFN-γ as described above. 146

Single cell preparations from subcutaneous injection site and lung. The subcutaneous 147


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tissues were collected at day 5 after subcutaneous inoculation of Histoplasma. A portion of the 148

tissue was snap frozen in liquid nitrogen. Another portion was cut into small pieces in 500 μl 149

of Dulbecco’s PBS (Biological Industries, Kibbutz Beit Haemek, Israel) by scissors and treated 150

with 5 mg/ml of Type I collagenase (Sigma-Aldrich) at 37°C for 1-1.5 h. The debris was 151

removed by filtration through nylon mesh. Lungs were harvested at day 4 after intratracheal 152

inoculation and ground between two frosted glass slides. Single cell suspension was obtained 153

by passing the suspension through cottons packed in Pasture pipette. 154

Immunofluorescence staining for confocal microscopy and flow cytometric analysis. 155

The subcutaneous tissues from the base of the tail where mice were immunized were 156

cryopreserved. The sections were fixed in acetone for 10 min and air-dried. The slides were 157

blocked with Dulbecco’s PBS (Biological Industries) containing 5% inactivated fetal calf 158

serum for 20 min, stained with PE-anti-F4/80, -anti-Gr-1, -anti-CD3 or FITC-anti-CD11c, 159

-anti-B220 mAbs at 4°C in humidified chamber in the dark overnight. After washing, Hoechst 160

33258 reagent was added and let stain for 3-5 min. The sections were viewed under confocal 161

microscope (Leica, Germany). The single cells isolated from subcutaneous tissue prepared as 162

described above were stained with FITC-anti-CD11c, PE-anti-F4/80 or PE-anti-Gr-1 at 4oC for 163

30 min. Cells were acquired by FACSCanto and analyzed with FACSDiva software program 164

(Becton Dickinson, San Diego, CA). 165


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TUNEL staining (In Situ Cell Death Detection). The cryosections were fixed with 4% 166

paraformaldehyde (Merck, Darmstadt, Germany) for 20 min. The sections were then treated 167

with 0.1% Triton X-100 (Sigma-Aldrich) at 4oC for 5 min and incubated with TUNEL reaction 168

mixture containing FITC-dNTP (Roche, Basel, Switzerland) at 37°C in the dark for 1 h. After 169

wash with PBS, the slides were stained with PE-anti-F4/80 or PE-anti-Gr-1. The sections were 170

examined under confocal microscope. 171

For flow cytometric analysis, cells isolated from subcutaneous tissues and lungs were first 172

stained with PE-anti-F4/80 or PE-anti-Gr-1 mAbs at 4oC for 30 min followed by addition of 173

TUNEL reaction mixture containing FITC-dNTP and incubated at 37°C in the dark for 1 h. 174

The cells were acquired by FACSCanto and analyzed with FACSDiva software program. 175

Preparation of and immunization with apoptotic phagocytes containing heat-killed 176

Histoplasma. Wild-type or β2m-/- mice were injected intraperitoneally with 4% thioglycollate 177

(DIFCO). To obtain macrophages, peritoneal cells were harvested 4 days later. The cells 178

(2×106) were allowed to adhere in RPMI 1640 medium overnight. Nonadherent cells were 179

removed by washing with pre-warmed HBSS. Twenty million heat-killed Histoplasma yeast 180

cells were added to the monolayer. After 2 h incubation at 37°C the monolayer was washed 181

with pre-warmed HBSS to remove free Histoplasma yeast cells. The culture was left at 37°C 182

overnight. Apoptosis was induced by treatment with 1 µg/ml LPS (Sigma-Aldrich) at 37°C for 183


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4 h, followed by addition of 5 mM ATP (Sigma-Aldrich) for another 45 min. The cells became 184

non-adherent. They were harvested by gentle pipeting and washed with RPMI 1640 medium 185

two times. One hundred percent of the cells were apoptotic as confirmed by TUNEL assay. 186

Apoptotic macrophages were suspended in 50 μl of RPMI 1640 medium and injected 187

subcutaneously into mice on two sides of the base of the tail (1×106 cells/injection) twice 2 188

weeks apart. 189

To obtain neutrophils, peritoneal cells were harvested 4 h after thioglycollate injection. 190

Seventy-five percent of the cells were Gr1+. Ten million of heat-killed Histoplamsa yeasts was 191

added to culture containing 1×106 neutrophils (MOI=10). After overnight incubation, the cells 192

were exposed to UV irradiation at 350 mJ/cm2 to induce apoptotic cell death (7). Ninety 193

percent of the cells were apoptotic after the treatment as confirmed by Annexin V (BD 194

Pharmingen) staining and flow cytometry analysis. Apoptotic cells were suspended in 50 μl 195

of RPMI 1640 medium and injected subcutaneously into mice on two sides of the base of the 196

tail (1×106 cells/injection) twice 2 weeks apart. 197

To prepare for CFSE-labeled apoptotic macrophages, thioglycollate-elicited peritoneal 198

macrophages were allowed to take up heat-killed Histoplasma as described previously. Before 199

induction of apoptosis, CFSE (Invitrogen, Carlsbad, CA) at final concentration of 5 μM was 200

added to the macrophage culture and let stand for 5 min. The monolayers were washed free of 201


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CFSE and treated with LPS and ATP to induce apoptosis. The detached cells were collected, 202

washed and injected subcutaneously into mice. 203

Injection of apoptosis inhibitor. Boc-D-FMK (pan-caspase inhibitor, 0.2 μmol, 204

Sigma-Aldrich) or Z-FA-FMK (control peptide, 0.2 μmol, Sigma-Aldrich) were dissolved in 205

10 μl DMSO for storage and diluted with 190 μl sterilized Dulbecco’s PBS immediately before 206

injection. Mice were injected intraperitoneally with Boc-D-FMK or Z-FA-FMK at the time of 207

intratracheal infection of Histoplasma and daily afterwards until the day of experiment (2). 208

In vivo cell depletion. To deplete CD11c+ dendritic cells, CD11c-DTR transgenic and 209

wild-type control mice were injected with 0.1 μg diphtheria toxin (in 200μl PBS, 210

Sigma-Aldrich) intraperitoneally at days -1 and 2 after immunization with [Apoptotic pMac 211

(HK Hc)]. Diphtheria toxin treatment depleted >86% of CD11c+ in the spleen as determined by 212

flow cytometry at 3 to 4 days after the last dose of diphtheria toxin. To deplete CD4+ and/or 213

CD8+ T cells, mice were injected intraperitoneally with concentrated supernatants from 214

hybridoma GK1.5 (anti-CD4) or 2.43 (anti-CD8) or both, one day before and twice weekly 215

after challenge with Histoplasma. The protein concentrations of concentrated hybridoma 216

supernatants were 4.0 mg of GK1.5 and/or 2.5 mg of 2.43 per injection. The treatment depleted 217

>95% of CD4+ and >95% of CD8+ T cells in the spleen as determined by flow cytometry at 3 218

to 4 days after the last dose of antibody treatment. 219


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Determination of fungal burden. Mice immunized subcutaneously with live Histoplasma, 220

[apoptotic pMac (HK Hc)], or RPMI 1640 medium were challenged intravenously with 221

2.5×104 live Histoplasma yeast cells at day 5 after second immunization. Spleens were 222

harvested and homogenized at day 10 after challenge; the homogenates were plated on 223

cysteine-blood-glucose agar and incubated at 30oC. Mycelial colonies were counted at 10-14 224

days after incubation. Fungal burdens in the spleens were expressed as log10 value of CFU per 225

spleen as previously described (14). 226

Lungs from mice treated with caspase inhibitor, control peptide or left untreated were 227

collected at day 10 after intratracheal inoculation of 2×105 Histoplasma yeast cells. The lungs 228

were homogenized and homogenates were plated on cysteine-blood-glucose agar and 229

incubated for 10-14 days. The CFU in the control mice was taken as 100% and the CFUs in the 230

experimental groups were compared to the control. The data were presented as percent change 231

of CFU. 232

H and E staining. The cryosections were fixed with 4% paraformaldehyde and stained with 233

0.1% hemotoxylin and 2% eosin for 5 min, respectively. 234

Statistics. The comparisons between multiple groups were analyzed by one way ANOVA 235

followed by the post hoc Tukey test, performed using the program Prism 4.0 (GraphPad 236

Software). The comparisons between two groups were analyzed by Student’s t test. The level 237


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of statistical significance was defined as P < 0.05. 238

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RESULTS 240

Apoptotic macrophages as antigen donors are efficient in delivering Histoplasma 241

antigen to induce functional CD8+ and CD4+ T cell activation in vivo. In previous in vitro 242

studies we showed that [Apoptotic pMac (HK Hc)] deliver fungal antigens contained within 243

them to dendritic cells, which in turn cross-present Histoplasma antigens to activate sensitized 244

CD8+ T cells (14). Here we extended the study and immunized mice with apoptotic 245

macrophages that had taken up Histoplasma yeasts. IFN-γ-production as determined by 246

intracellular cytokine staining was used as a readout for functional activation of both CD8+ and 247

CD4+ T cells (13). Results in Fig. 1A show that [Apoptotic pMac (HK Hc)] at 2 × 106 and 4 248

× 106 efficiently induced functionally activated CD8+ and CD4+ T cells. Although the numbers 249

of IFN-γ-producing CD8+ and CD4+ T cells did not reach the levels as in the mice receiving 250

live Histoplasma, they were significantly higher than that of mice receiving heat-killed 251

Histoplasma alone (Fig. 1A) (P < 0.001 for CD8+ and P < 0.05 for CD4+). Moreover, CD8+ T 252

cells not only expressed IFN-γ but also granzyme B (Fig. 1B). These results indicate that 253

heat-killed Histoplasma antigens contained within apoptotic macrophages are efficiently 254

delivered to cross-prime CD8+ T cells and activate CD4+ T cells in vivo. 255

Fig. 2A further revealed that immunization with apoptotic macrophages mixed with 256

heat-killed Histoplasma [Apoptotic pMac + HK Hc], viable macrophages with ingested 257


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heat-killed Histoplasma [pMac (HK Hc)], apoptotic macrophages [Apoptotic pMac] or viable 258

macrophages [pMac] alone did not induce T cell responses at a level comparable to that of 259

immunization with [Apoptotic pMac (HK Hc)]. It is noteworthy that apoptotic pMac simply 260

mixed with heat-killed Histoplasma before injection [Apoptotic pMac + HK Hc] did not 261

induce a strong CD8+ or CD4+ T cell response (Fig. 2A), suggesting that Histoplasma antigens 262

need to be contained within apoptotic macrophages for antigen presentation. Moreover, 263

immunization with apoptotic neutrophils containing Histoplasma [Apoptotic pNeu (HK Hc)] 264

was as efficient as [Apoptotic pMac (HK Hc)] in inducing a strong CD8+ or CD4+ T cell 265

response (Fig. 2B), which indicates that after Histoplasma yeasts are taken up by the 266

phagocytes, the fungal antigen contained within the phagocytes can be presented to activate 267

CD8+ and CD4+ T cells. Interestingly, immunizing mice with [Apoptotic pMac (HK Hc)] that 268

lack MHC class I molecules (β2m-/-) activated CD8+ T cells as efficiently as wild-type 269

macrophages did (Fig. 2C), indicating that apoptotic cells serve as antigen donor cells to 270

deliver Histoplasma antigens rather than as antigen-presenting cells in the immunized host. 271

CD8+ and CD4+ T cells have equal contribution to host defense after immunization 272

with apoptotic macrophages containing heat-killed Histopalsma. Next, mice immunized 273

with [Apoptotic pMac (HK Hc)] were challenged with Histoplasma intravenously. Fig. 3A 274

shows that immunized mice had significantly lower fungal burden in the spleen compared with 275


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unimmunized mice (CM, P < 0.01) at day 10 after challenge. Depleting CD8+ and CD4+ T 276

cells singly in mice immunized with [apoptotic pMac (HK Hc)] at the time of challenge 277

significantly increased fungal burden by 61-fold and 74-fold, respectively, and depleting both 278

populations increased fungal burden by 490-fold (Fig. 3B). These results together with those in 279

Figs. 1 and 2 demonstrate that immunization with [Apoptotic pMac (HK Hc)] effectively 280

induces protective CD8+ cells and the contribution of CD8+ T cells to host defense against 281

histoplasmosis is comparable to CD4+ T cells. 282

Reduction of macrophage apoptosis due to iNOS deficiency decreases CD8+ T cell 283

activation. To explore the relationship between macrophage cell death and CD8+ T cell 284

activation, iNOS-/- and wild-type mice were infected with Histoplasma intratracheally. Fig. 4A 285

shows that the number of apoptotic F4/80+ cells in the lungs of infected iNOS-/- mice was 286

significantly lower than in the wild-type mice (P < 0.05). Coinciding with lower number of 287

apoptotic F4/80+ cells in the lungs, the percentage of IFN-γ-producing CD8+ T cells was also 288

significantly lower in iNOS-/- mice than in wild-type mice (Fig. 4B) (P < 0.05). Interestingly, 289

CD4+ T cell response in iNOS-/- mice was not affected by the reduction of apoptotic F4/80+ 290

cells (Fig. 4B). Results of macrophage phagocytosis assay and stimulation of CD8+ T cells 291

with anti-CD3 and anti-CD28 antibodies showed that macrophages and CD8+ T cells from 292

iNOS-/- mice were functionally comparable to that of wild-type mice (data not shown). 293


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Additionally, bone marrow-derived dendritic cells from iNOS-/- mice expressed higher MHC I, 294

CD40 but lower MHC II molecules on the surface than cells from the wild-type mice after 295

infection with Histoplasma yeasts (data not shown). These observations excluded the 296

possibility of the intrinsic defect of macrophages or the antigen-presentation functions of 297

dendritic cells in the iNOS-/- mice affecting CD8+ T cell activation. These results together 298

indicated that iNOS, possibly through production of nitric oxide, is the cause of macrophage 299

apoptotic cell death and that macrophage death is important to CD8+ T cell activation in 300

pulmonary infection by Histoplasma. 301

Inhibition of apoptotic cell death by caspase inhibitor decreases CD8+ T cell response 302

and exacerbates pulmonary histoplasmosis. To further demonstrate the importance of 303

apoptosis of antigen donor cells in activation of CD8+ T cell response after pulmonary 304

infection, we treated infected mice with caspase inhibitor Boc-D-FMK (26). The number of 305

apoptotic F4/80+ cells at early phase after infection was significantly reduced in mice treated 306

with Boc-D-FMK compared with treatment with control peptide Z-FA-FMK (23) (Fig. 5A) (P 307

< 0.05). Interestingly, in consistent with what we observed in iNOS-/- mice, CD8+ T cell but not 308

CD4+ T cell response was reduced in mice whose macrophage apoptosis was inhibited (Fig. 309

5B). Notably, Boc-D-FMK treatment resulted in higher fungal burden than Z-FA-FMK 310

treatment (Fig. 5C). These results together indicate that macrophage apoptosis is critical to 311


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CD8+ T cell activation and subsequent fungal clearance in Histoplasma infection. 312

CD8+ T cell response in mice subcutaneously immunized with viable Histoplasma 313

yeasts correlates with infiltrating cell death. It was reported that subcutaneous immunization 314

with live Histoplasma yeast cells elicits protective CD8+ T cell response even in the absence of 315

CD4+ T cells (32). Whether induction of protective CD8+ T cell response correlates with 316

antigen donor cell death in this mouse model was a question to be addressed. 317

Immunofluorescence analysis revealed that the cellular infiltrates in the subcutaneous tissues 318

(Fig. 6A) were mostly Gr-1+ and F4/80+ cells, some CD11c+ cells and very few B220+ and 319

CD3+ cells (Fig. 6 B and 6C). Single cell suspension prepared from the digested subcutaneous 320

tissues contained mainly Gr-1+ (68.9%) and F4/80+ (18.9%) cells (Fig. 6B), confirming that 321

granulocytes and macrophages are the major cell populations at the site of subcutaneous 322

inoculation. It is noteworthy that both infiltrating granulocytes and macrophages became 323

apoptotic (Fig. 6D) and granulocytes and macrophages constituted 77.6% and 17.2%, 324

respectively, of the total apoptotic cell populations at day 5 after inoculation (Fig. 6E). 325

Moreover, subcutaneous inoculation of iNOS-/- mice with viable Histoplasma induced CD4+ T 326

cell but not CD8+ T cell activation (Fig. 6F). Together, these results point to the importance of 327

apoptotic cell death during the early phase of Histoplasma infection in inducing protective 328

CD8+ T cell responses and fungal clearance. 329


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CD11c+ cells are required for CD8+ T cell cross-priming. We further investigated the role 330

of host dendritic cells in presenting antigens contained within apoptotic macrophages to 331

activate CD8+ T cells. Mice were subcutaneously immunized with CFSE-labeled [Apoptotic 332

pMac (HK Hc)]. Five days after immunization, CFSE-labeled apoptotic macrophage 333

cytoplasmic materials were found inside of CD11c+ cells in the draining lymph nodes (Fig. 334

7A). Furthermore, depleting CD11c+ cells in CD11c-DTR transgenic mice by diphtheria toxin 335

treatment before inoculation of [Apoptotic pMac (HK Hc)] significantly reduced the 336

percentage of IFN-γ-producing CD8+ by 6.5-fold as compared with that of wild-type mice 337

treated with diphtheria toxin (Fig. 7B). These results indicate that host CD11c+ dendritic cells 338

at the inoculation site take up the fungal antigens contained within the apoptotic macrophages 339

after immunization with [Apoptotic pMac (HK Hc)] and migrate to the draining lymph nodes 340

to cross-prime CD8 T cells. 341

342


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DISCUSSION 343

Histoplasma capsulatum is a facultative intracellular fungal pathogen of the macrophage 344

(16). It has long been recognized that macrophage serves as the host cell as well as the effector 345

cell for Histoplasma yeast (17, 30, 31). In in vitro studies we showed that ingestion of 346

Histoplasma yeasts triggers macrophage apoptotic cell death and the apoptotic macrophage 347

serves as Histoplasma antigen donor to cross-prime CD8+ T cells (14). In this study, we 348

showed that immunization with [Apoptotic pMac (HK Hc)] or [Apoptotic pNeu (HK Hc)] 349

activate both CD8+ and CD4+ T cells. Remarkably, immunization with apoptotic macrophages 350

or neutrophils mixed with killed yeasts ([Apoptotic pMac + HK Hc] or [Apoptotic pNeu + HK 351

Hc]), or with viable macrophages containing killed yeasts [pMac (HK Hc)] does not efficiently 352

activate CD8+ and CD4+ T cells (Fig. 2A and 2B). In infection by Histoplasma, iNOS 353

deficiency and apoptosis inhibitor treatment decrease the number of apoptotic macrophages 354

and weaken CD8+ T cell but not CD4+ T cell response. The results of our study demonstrate 355

that apoptosis of phagocytes is an immune mechanism that is of critical importance to CD8+ T 356

cell activation in histoplasmosis. 357

Infection-induced apoptosis is a newly recognized immune function important to 358

anti-microbial immunity, especially in defense against phagosome-enclosed pathogens (21, 27, 359

33). Different host and fungal factors are known to be involved in Histoplasma-induced 360


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macrophage death. Our unpublished data showed that Histoplasma yeasts induced macrophage 361

iNOS expression. Coinciding with these results, we showed in this study that Histoplasma 362

infection-induced macrophage apoptosis is significantly reduced in iNOS-/- mice compared to 363

wild-type mice (Fig. 4). These findings together demonstrate that iNOS induction contributes 364

to macrophage death. On the fungus side, both the yeast cell wall component α-(1, 3)-glucan 365

and the secreted calcium-binding protein (CBP) are identified to be the virulence factors of 366

Histoplasma (19). Silencing α-(1, 3)-glucan biosynthesis reduces the ability of Histoplasma 367

yeast to cause macrophage-like P388D1 cell death (18) and the cbp1-null mutant lost the 368

ability to kill P388D1 cells in in vitro culture (11). Although it is not clear whether 369

Histoplasma yeast cell α-(1, 3)-glucan and secreted CBP cause macrophage to produce nitric 370

oxide, it appears that a built-in system exists in the interaction between the fungus and 371

macrophage to ensure macrophage death, which we showed is essential to CD8+ T cell 372

cross-priming. 373

We showed in this study that macrophages and granulocytes in the subcutaneous tissues as 374

well as macrophages in the lungs constitute the major cell populations during the early phase 375

of subcutaneous and pulmonary infection, respectively, and undergo apoptosis (Fig. 6 and 4). 376

The percentages of infiltrating CD3+ T cells, on the contrary, are low at the early time points 377

(data not shown). Allen and Deepe analyzed the apoptotic cell populations in the lungs of 378


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Histoplasma-infected mice at days 7, 14 and 21 after infection (2), the activation and 379

contraction phases of T cell response (13), and found that apoptotic CD3+ T cells were the 380

major apoptotic cells (2, 13). Their study showed that inhibition of apoptosis associates with 381

elevation of IL-4 and IL-10 and the release of these cytokines exacerbates the severity of 382

infection. We found that administration of Boc-D-FMK beginning at the time of infection 383

reduces the number of apoptotic macrophages by about 50% at day 4 and dampens the 384

subsequent CD8+ T cell but not CD4+ T cell response (Fig. 5A and 5B). This causal 385

relationship demonstrates that macrophage apoptotic cell death at the early phase of infection, 386

the time when antigen-processing, presentation and cross-presentation take place, modulates 387

the protective CD8+ T cell response. Our study and that of Allen and Deepe together stress the 388

importance of apoptosis at the antigen processing and presentation phases as well as the T cell 389

contraction phase is critical to protective immune response in histoplasmosis. 390

It has been reported that immunization with apoptotic vesicles prepared from 391

BCG-OVA-infected macrophages induces adoptively transferred OT-1 CD8+ T cell activation 392

and mice are protected against challenge by Mycobacterium tuberculosis (28). Injection of 393

apoptotic antigen-pulsed dendritic cells induces both humoral and cellular immune responses 394

and confers protection against oral challenge of Toxoplasma gondii (4). In this study, we 395

showed that immunization with [Apoptotic pMac (HK Hc)] induces functional CD8+ and 396


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CD4+ T cells and both of them contribute equally to efficient clearance of Histoplasma. These 397

results together suggest that apoptotic cells delivering antigens to elicit functional and 398

protective CD8+ and CD4+ T cell responses can be used to immunize against intracellular 399

bacterial, parasitic as well as fungal pathogens. 400

After subcutaneous injection of live Histoplasma, there is massive granulocyte infiltration 401

and 60-80% of the cells are apoptotic at days 3-7 after inoculation (Fig. 6A-6E). Apoptotic 402

neutrophils are as efficient as apoptotic macrophages in delivering Histoplasma antigens to 403

activate CD8+ and CD4+ T cells (Fig. 2B). Granulocyte infiltration in the spleen after systemic 404

infection or in the lungs after intratracheal infection is not as pronounced as it is in the 405

subcutaneous tissues after local infection (personal observation). Their contribution to 406

cross-presentation in infection through the natural routes may not be as important as the 407

macrophages. However, utilizing apoptotic granulocytes/neutrophils as antigen donors for 408

immunization is also a feasible option. 409

In summary, we showed that phagocyte apoptosis during the early phase of Histoplasma 410

infection is important to CD8+ T cell functional activation. Immunization with [Apoptotic 411

pMac (HK Hc)] or alternatively [Apoptotic pNeu + HK Hc] activates both CD8+ and CD4+ T 412

cells and protects against fungal challenge. Thus, exploiting the role of apoptotic phagocytes as 413

antigen donor cell is a viable strategy to develop efficacious vaccines against histoplasmosis. 414


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ACKNOWLEDGEMENTS 415

This work was supported by National Science Council grants NSC 94-2320-B-002-064, 416

95-2320-B-002-027 and 96-2320-B-002-011 to B.W.H., National Science Council Fellowship 417

grants NSC 94-2811-B-002-076, 95-2811-B-002-073, and 96-2811-B-002-069 to J.S.L., and 418

Southern Taiwan Science Park grant EY13151099 to C.L.C.. 419

We acknowledge the support provided by the Seventh Core Lab of the Department of 420

Medical Research of the National Taiwan University Hospital. 421

422


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31. Wu-Hsieh, B. A., and D. H. Howard. 1987. Inhibition of the intracellular growth 513

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2003. Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 517

help in exogenous antigen presentation to CD8+ T cells: implications for vaccine 518

development in immune-deficient hosts. The Journal of experimental medicine 519

197:1405-1416. 520

33. Yrlid, U., and M. J. Wick. 2000. Salmonella-induced apoptosis of infected 521

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524


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FIGURE LEGENDS 525

FIG. 1. Immunization with apoptotic macrophages containing Histoplasma induces CD8+ 526

and CD4+ T cell activation. Thioglycollate-elicited peritoneal macrophages were allowed to 527

ingest heat-killed Histoplasma before treatment with LPS and ATP to induce apoptosis. Mice 528

were inoculated subcutaneously with live Histoplasma (Live Hc, 2 × 106), heat-killed 529

Histoplasma (HK Hc, 2×106) or apoptotic macrophages containing heat-killed Histoplasma 530

([Apoptotic pMac (HK Hc)], 4, 2, 1, or 0.2×106) on days -14 and 0. Control mice were given 531

medium (CM) alone. At day 5, cells were isolated from inguinal lymph nodes and stimulated 532

with heat-killed Histoplasma for 24 h. (A) Lymph node cells were stained with FITC-anti-CD4, 533

APC-anti-CD8 and PE-anti-IFN-γ antibodies and analyzed by flow cytometry. Bar graphs 534

indicate the percentages of IFN-γ+CD8+ or IFN-γ+CD4+ T cells in the total CD8+ or CD4+ T 535

cell population. The data shown are the mean ± SD of a total of 4 mice used in 3 separate 536

experiments, and the data from all 4 mice are presented. The P values were obtained by 537

comparing the percentages of IFN-γ-producing cells in the mice receiving HK Hc and 4 538

different doses of [Apoptotic pMac (HK Hc)] by post hoc Tukey test. (*, P < 0.05; **, P < 539

0.01; ****, P < 0.001; NS, not significant). (B) Lymph node cells were stained with 540

APC-anti-CD8 and PE-anti-granzyme B (grB) antibodies or PE-mouse IgG1 as isotype control. 541

The percentages of granzyme B-producing CD8+ T cells in the total CD8+ cell population were 542


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analyzed (*, P < 0.05; NS, not significant). The data shown are mean ± SD of 5 mice from 3 543

independent experiments. 544

FIG. 2. Apoptotic phagocytes containing Histoplasma induce T cell response independent 545

of donor cell MHC class I. (A) Thioglycollate-elicited peritoneal macrophages were allowed to 546

ingest heat-killed Histoplasma before treatment with LPS and ATP. Mice were subcutaneously 547

inoculated with 2×106 of apoptotic [Apoptotic pMac (HK Hc)] or nonapoptotic [pMac (HK 548

Hc)] pMac containing heat-killed Histoplasma, apoptotic [Apoptotic pMac] or nonapoptotic 549

[pMac] pMac without ingested heat-killed Histoplasma, or apoptotic pMac mixed with 550

heat-killed Histoplasma [Apoptotic pMac + HK Hc] at days -14 and 0. Cells from inguinal 551

lymph nodes were harvested at day 5 and stained with FITC-anti-CD4, APC-anti-CD8 and 552

PE-anti-IFN-γ antibodies. IFN-γ-producing CD8+ and CD4+ T cells were analyzed by flow 553

cytometry. Data shown are the mean ± SD of 4 mice from 3 independent experiments. The P 554

values were obtained by comparing the percentages of IFN-γ-producing cells in the five groups 555

by post hoc Tukey test. (*, P <0.05; **, P <0.01; ****, P <0.001). (B) Peritoneal macrophages 556

(pMac) or neutrophils (pNeu) were allowed to ingest heat-killed Histoplasma [pMac (HK Hc) 557

or pNeu (HK Hc)] or mix with heat-killed Histoplasma [pMac+HK Hc or pNeu+HK Hc] 558

before inducing apoptosis by LPS and ATP treatment or UV irradiation. Mice were 559

subcutaneously inoculated with these apoptotic cells as described in (A) and the CD8+ and 560


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CD4+ T cell responses elicited were analyzed. Mice inoculated with PBS or live Hc were 561

served as experimental controls. Data shown are the mean ± SD of 4 mice from 2 562

independent experiments. The P values were obtained by comparing the percentages of 563

IFN-γ-producing cells in the four groups of mice receiving apoptotic cells by post hoc Tukey 564

test. (*, P <0.05; NS, not significant). (C) Peritoneal macrophages from wild-type (WT, ■) or 565

β2 microglobulin-deficient (β2m-/-, □) mice were allowed to ingest heat-killed Histoplasma 566

before treatment with LPS and ATP. Wild-type mice were immunized with [Apoptotic pMac 567

(HK Hc)] that obtained from either wild-type or β2 microglobulin-deficient mice. The 568

immunization schedule, time of experiment and staining were the same as described in (A). 569

Cells harvested from mice receiving wild-type macrophages cultured in medium only (CM) 570

served as controls. Data shown are the mean ± SD of 4 mice from 3 independent experiments. 571

(****, P <0.001; NS, not significant). 572

FIG. 3. Immunization with apoptotic macrophages containing Histoplasma confers CD8+ 573

and CD4+ T cell-dependent protection against challenge. (A) Wild-type mice were inoculated 574

subcutaneously with live Histoplasma (2×106 yeast cells/mouse), apoptotic macrophages 575

containing heat-killed Histoplasma ([Apoptotic pMac (HK Hc)], 2×106 cells/mouse) or 576

medium (CM) as described in Fig. 2A. At day 5, mice were challenged intravenously with 577

2.5×104 live Histoplasma yeasts. Fungal burden in the spleen was assessed 10 days after 578


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challenge. (B) Wild-type mice were inoculated subcutaneously with [Apoptotic pMac (HK Hc)] 579

as described in (A). At the day before challenge and twice weekly thereafter, mice were treated 580

with depleting antibodies against either CD4 or CD8, or both. Fungal burden in the spleen was 581

assessed 10 days after challenge. (A and B) Data represent the mean ± SD of 5-6 mice per 582

group. The P values were obtained by comparing the results by post hoc Tukey test (**, P 583

<0.01; ****, P <0.001; NS, not significant). 584

FIG. 4. iNOS deficiency reduces F4/80+ cell apoptosis and weakens CD8+ T cell response in 585

pulmonary histoplasmosis. Wild-type (■) and iNOS-/- (□) mice were infected intratracheally 586

with 2×105 live Histoplasma. (A) At day 5 after infection, lung cells were isolated and stained 587

with PE-anti-F4/80 antibody and TUNEL reagents containing FITC-dNTP. F4/80+TUNEL+ 588

apoptotic cells were analyzed by flow cytometry. Data shown are the mean ± SD of the total 589

number of apoptotic F4/80+ cells of 3 mice from 3 independent experiments. (B) At day 10 590

after infection, the mediastinal lymph nodes were harvested and cells were stained with 591

PE-anti-IFN-γ and FITC-anti-CD4 or APC-anti-CD8 antibodies. Data shown are the mean ± 592

SD of the percentages of IFN-γ-producing CD8+ or CD4+ T cells in the total CD8+ or CD4+ T 593

cell population of 6 mice from 3 independent experiments. Cells harvested from uninfected 594

wild-type mice served as controls. The P values were obtained by comparing the results of two 595

groups linked by a bracket using Student’s t test (*, P <0.05; NS, not significant). 596


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FIG. 5. Inhibition of F4/80+ cell apoptosis reduces CD8+ T cell response and increases 597

fungal burden. Wild-type mice were treated daily with apoptosis inhibitor Boc-D-FMK, 598

control peptide Z-FA-FMK or left untreated starting at the day of intratracheal inoculation of 599

2×105 live Histoplasma. (A) Lung cells isolated from infected mice at day 4 after infection 600

were stained with TUNEL reagents containing FITC-dNTP and PE-anti-F4/80 antibodies. 601

Apoptotic F4/80+ cells in the lungs were analyzed by flow cytometry. Data shown are the 602

mean ± SD of the total number of F4/80+ cells of 3 mice from 3 independent experiments (*, P 603

<0.05; NS, not significant, by Student’s t test). (B) Mediastinal lymph node cells were 604

harvested at day 10 after infection. Cells were stained with PE-anti-IFN-γ and APC-anti-CD8 605

or FITC-anti-CD4 antibodies and analyzed by flow cytometry. Data shown are the mean ± SD 606

of the percentages of IFN-γ-producing CD8+ and CD4+ cells in the total CD8+ or CD4+ T cell 607

population of 5 mice from 4 independent experiments (**, P <0.01; NS, not significant by 608

Student’s t test). (C) Fungal burdens in the lungs of infected mice were assessed at day 10 after 609

intratracheal infection. The CFU from infected mice treated with Z-FA-FMK and that treated 610

with Boc-D-FMK were compared against CFU from infected mice without treatment. The data 611

are presented as percent change of CFU and shown as the mean + SD of total 5-6 mice per 612

group (***, P<0.005 by Student’s t test). 613

FIG. 6. CD8+ T cell response in mice subcutaneously immunized with Histoplasma 614


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correlates with infiltrating cell death. (A-E) Wild-type mice were injected subcutaneously with 615

live Histoplasma at day 0 and subcutaneous tissues were collected at day 5. (A) The 616

subcutaneous tissues at the inoculation site were harvested. The tissues were cryosectioned and 617

stained with H&E (magnification 40×). The circle showed cellular infiltration in the 618

inoculation site. (B) Subcutaneous tissues of the inoculation sites were treated with type I 619

collagenase to obtain single cells. Isolated cells were stained with FITC-anti-CD11c and 620

PE-anti-Gr-1 or PE-anti-F4/80 antibodies and analyzed by flow cytometry. The numbers 621

indicate the percentages of CD11c+, Gr-1+ or F4/80+ cells in the total cell population. The data 622

shown are one representative mouse of two independent experiments. (C) Cryosections of the 623

subcutaneous tissues at the inoculation site were prepared and the cell populations were 624

determined by staining with PE-anti-Gr-1, -F4/80, or -CD3 (red) and FITC-anti-CD11c, or 625

-B220 (green) antibodies and Hoechst stain 33258 (blue). Images were viewed under confocol 626

microscope. The length of the white lines is 40μm (magnification, 630×). (D) Cryosections of 627

the subcutaneous tissues at inoculation site were stained with TUNEL reagents containing 628

FITC-dNTP and PE-anti-Gr-1 or PE-anti-F4/80 antibodies. Images were viewed under 629

confocal microscope. The arrowheads point to apoptotic granulocytes or macrophages where 630

TUNEL reactive nuclei are adjacent to Gr-1+ or F4/80+ staining. The length of the white lines 631

is 40μm (magnification, 630×). (E) Cells isolated from subcutaneous tissues at days 3, 5, and 7 632


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after inoculation of Histoplasma were stained with TUNEL reagents and PE-anti-F4/80 or 633

PE-anti-Gr-1 antibodies. The percentages of apoptotic macrophages (TUNEL+F4/80+) and 634

granulocytes (TUNEL+Gr-1+) were analyzed by flow cytometry. The bar graphs show the 635

mean ± SD of the percentages of apoptotic macrophages or granulocytes in the total TUNEL+ 636

apoptotic cells. (F) Wild-type (■) and iNOS-/- (□) mice were injected subcutaneously with live 637

Histoplasma at days -14 and 0. Cells were isolated and stained as described in Fig. 1. Data 638

shown are the mean ± SD of 6 mice of 3 independent experiments (***, P <0.005; NS, not 639

significant, by Student’s t test). 640

FIG. 7. CD11c+ cells are critical to CD8+ T cell activation after apoptotic macrophages 641

containing Histoplasma immunization. (A) CFSE-labeled peritoneal macrophages were 642

allowed to ingest heat-killed Histoplasma before LPS and ATP treatment. Wild-type mice 643

were inoculated subcutaneously with [Apoptotic pMac (HK Hc)]. Five days after inoculation, 644

the inguinal lymph nodes were snap frozen and cryosectioned. The sections were stained with 645

PE-anti-CD11c mAb and viewed under confocal microscope. The arrowheads point to 646

CFSE-labeled macrophage cytoplasmic materials that were contained within CD11c+ cells. (B) 647

Wild-type mice (WT, ■) and CD11c-DTR transgenic mice (CD11c-DTR, □) were given 100 648

ng of diphtheria toxin intraperitonealy at days -1 and 2 and inoculated subcutaneously with 649

[Apoptotic pMac (HK Hc)] at day 0. Draining lymph node cells were harvested at day 5 and 650


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stimulated with suboptimal concentrations of plate-bound anti-CD3 (0.5μg/ml) and anti-CD28 651

(0.5μg/ml) antibodies for 5 h. IFN-γ-producing CD8+ T cells were analyzed by flow cytometry. 652

Data shown are the mean ± SD of 4 mice from 3 independent experiments. Lymph node cells 653

from wild-type mice inoculated with medium only (CM) served as controls. (***, P <0.005, by 654

Student’s t test). 655

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