PD-1 deficiency Enhances Humoral Immunity of Malaria infection

Loading...
IAI Accepted Manuscript Posted Online 2 March 2015 Infect. Immun. doi:10.1128/IAI.02621-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

PD-1 deficiency Enhances Humoral Immunity of Malaria

2

infection-treated vaccine

3

Taiping Liu1, Xiao Lu1, Chenghao Zhao1, Xiaolan Fu2, Tingting Zhao2#, Wenyue Xu1#

5

1

6

R. China.

7

2

8

China.

Department of Pathogenic Biology, Third Military Medical University, Chongqing, P.

Institute of Immunology of PLA, Third Military Medical University, Chongqing, P. R.

9 10

Running title: PD-1 deficiency and humoral immunity

11

These authors declare that there is no conflict of interest.

12 13 14 15 16 17 18 19 20

Correspondence: Wenyue Xu, Department of Pathogenic Biology, Third Military Medical University, Chongqing, P. R. China, Tel: 86 23 68752236, Fax: 86 23 68752236, e-mail: [email protected]; or Tingting Zhao, Institute of Immunology of PLA, Third Military Medical University, Chongqing, P.R. China. E-mail: [email protected] #

1

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

4

21

Abstract Malaria infection-treatment vaccine (ITV) is a promising strategy to induce

23

homologous and heterologous protective immunity against the blood stage of the

24

parasite. However, the underlying mechanism of protection remains largely unknown.

25

Here, we found that a malaria-specific Ab could mediate the protective immunity of

26

the ITV-immunized mice. Interestingly, PD-1 deficiency greatly elevated the levels of

27

both malaria-specific total IgG and subclass IgG2a and enhanced the protective

28

efficacy of ITV-immunized mice against the blood-stage challenge. A serum adoptive

29

transfer assay demonstrated that the increased Ab level contributed to the enhanced

30

protective efficacy of the immunized PD-1-deficient mice. Further study showed that

31

PD-1 deficiency could also promote the expansion of germinal center (GC) B cells

32

and malaria parasite-specific TFH cells in the spleens of the ITV-immunized mice.

33

These results implicate that PD-1 deficiency improves the protective efficacy of

34

ITV-immunized mice by promoting the generation of malaria parasite-specific Ab and

35

the expansion of GC B cells. The results of this study provide us with new evidence to

36

support the negative function PD-1 on humoral immunity and will guide the design of

37

a more effective malaria vaccine.

38 39 40 41 42 2

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

22

43

Introduction

44

Although malaria control programs have led to an extensive reduction in malaria

45

incidence and mortality, it remains one of the most threatening diseases worldwide. It

46

is estimated that 207 million cases and 627,000 malaria deaths occurred in 2012 (1). A vaccine is regarded as the most cost-effective strategy to prevent malaria

48

infection (2). Most malaria subunit blood-stage vaccines have been designed to induce

49

antibodies (Ab) against a variety of surface proteins on the merozoite to block the

50

invasion of red blood cells (3). However, the invasion of the merozoites into red blood

51

cells is controlled by multiple redundant proteins (4), and Ab against one or two

52

merozoite surface proteins are unable to effectively prevent the infection of red blood

53

cells with the malaria parasite (4). Furthermore, most merozoite surface proteins

54

exhibit antigenic polymorphism under selective pressure (5). To date, there is no

55

malaria subunit vaccine available worldwide.

56

In

contrast

to

the

subunit

malaria

vaccine,

the

malaria

57

infection-treatment-vaccination (ITV), which involves infection with live malaria

58

parasites under curative anti-malarial drug coverage, has been reported to induce

59

antibodies specific for the merozoite surface antigens conserved between

60

heterologous strains but not for the variant surface antigens (6). ITV induces strong

61

protective immunity against the blood-stage of the parasite in animals (7) and humans

62

(8). Interestingly, ITV can also confer cross-protection against the liver stage of

63

malaria by inducing cellular immune responses (7). However, the underlying

64

mechanism of protective immunity induced by ITV is still largely unknown. 3

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

47

Follicular helper CD4 T(TFH) cells are characterized by the high expression of

66

chemokine receptor CXCR5, programmed death 1 (PD-1), lineage-specific

67

transcription regulator Bcl6, SAP (SH2D1A), IL-21, and ICOS and are recognized as

68

specialized providers of cognate B cell help (9). Of these characteristic molecules,

69

PD-1 has been reported to provide modulatory signals to GC TFH cells, but its

70

function in the modulation of humoral immunity remains unresolved. Some evidence

71

has shown that the blockade of PD-L1 or PD-1 reinforces TFH cell expansion,

72

increases the number of GC B cells and plasmablasts and enhances antigen-specific

73

Ab responses (10, 11). However, attenuated humoral immune responses also have

74

been observed after blockade of PD-1 signaling (12-14). Therefore, the exact role of

75

PD-1 signaling in the protective immunity of the ITV-immunized mice remains

76

unclear.

77

In this study, we found that PD-1-deficiency greatly improved the protective

78

efficacy of ITV-immunized mice against a malaria blood stage challenge. This

79

phenomenon was attributed to the elevated malaria parasite-specific Ab in the

80

immunized PD-1-deficient mice. Additionally, we also observed increased GC B cells

81

and the expansion of TFH cells in the immunized PD-1-deficient mice. Thus, our data

82

further confirmed the negative effect of PD-1 signaling on humoral immunity and

83

shed new light on the design of effective malaria vaccine.

84 85 86 4

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

65

87

Materials and Methods

88

Mice and Plasmodium parasites PD-1-/- mice (BALB/c background) were obtained from the Jackson Laboratory

90

(Bar Harbor, ME). Specific pathogen-free BALB/c mice, at 6-8 week of age, were

91

purchased from the Beijing Animal Institute. All animal protocols were reviewed and

92

approved by the Animal Ethics Committee of the Third Military Medical University

93

Institute of Medical Research. The lethal strain Plasmodium yoelii 17XL was obtained

94

from MR4 (Malaria Research and Reference Reagent Resource Center, Manassas,

95

Virginia) and maintained by i.p. passages in mice.

96 97

Immunization and challenge

98

The immunization schedule was performed as previously described (7) with minor

99

modifications. Briefly, mice were i.v. injected with 1×106 P. yoelii 17XL live iRBC

100

(Py-iRBC) or an equivalent amount of normal RBC with or without 100 µl of 8

101

mg/ml chloroquine (CQ, Sigma-Aldrich) diluted in saline daily for 15 days starting

102

from the day of iRBC injection. The mice were maintained for 21 days after the last

103

CQ injection to allow complete elimination of the drug and challenged i.p. with 1×106

104

Py-iRBC.

105 106

Adoptive serum transfer assay and CD4+T cell depletion

107

For serum transfer, naïve BALB/c mice were i.v. injected on days -1, 0 and 1 with

108

0.2 ml naïve mice serum, ITV-immunized WT or PD-1-/- mice serum collected 21 days 5

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

89

after the last CQ injection, as described previously (15). The mice were challenged

110

with 2.5×104 Py-iRBC on day 0, and parasitemia and the survival rate were

111

determined. For CD4 depletion studies, an anti-CD4-depleting mAb (GK1.5 clone,

112

200 µg per mouse in 200 µl of PBS; BioXcell) or control Ab was i.p. injected on days

113

-1 and 1 (20 and 22 days after the last CQ injection) according to a previously

114

described protocol(16). CD4+ T cell depletion was verified by staining blood samples

115

with anti-CD4 (clone RM4-5, eBioscience). The mice were then challenged with

116

1×106 Py-iRBC on day 0.

117 118

Detection of malaria-specific IgG in serum

119

The serum was collected from the naïve mice, ITV-immunized WT and PD-1-/-

120

mice at 21 days after the last CQ injection. Hyperimmune sera (HIS) were collected

121

from the ITV-immunized WT mice that had recovered from the P. yoelii 17XL

122

infection. The malaria-specific total IgG, IgG1 and IgG2a in the serum were detected

123

as previously described (15, 17). Briefly, P. yoelii 17XL-infected mouse blood was

124

collected, lysed with 0.01% saponin (Sigma-Aldrich) at 37°C for 20 min, and

125

sonicated in PBS. NuncMaxiSorp Immunoplates (NalgeNunc) were coated with

126

parasite Ag at a concentration of 5-10 μg/ml overnight at 4°C and co-incubated with

127

serial dilutions of sera from the ITV-immunized WT and PD-1-/- mice.

128

Biotin-conjugated anti-mouse IgG1 and IgG2a (Biolegend) were added to the plates

129

to detect IgG1 and IgG2a. After washing with wash buffer, the plates were incubated

130

with HRP-conjugated anti-mouse IgG or HRP-conjugated streptavidin (Biolegend), 6

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

109

131

and 3,3’,5,5’-tetramethylbenzidine was added (Biolegend). The absorbance at wave

132

length of 450 nm was read using a spectrophotometer.

133 134

Flow cytometry analysis of GC B and Malaria-specific TFH cells Both GC B and malaria-specific TFH cells from the naïve mice, ITV-immunized

136

WT and PD-1-/- mice were analyzed at 7, 9, 11, 14 and 21 days (at 21, 23, 25, 28 and

137

35 days after the start of CQ treatment), respectively, after the last CQ injection. In

138

brief, single-cell suspensions of splenocytes were prepared and washed in flow

139

cytometry buffer (PBS with 2% FCS and 0.05% sodium azide) followed by blocking

140

with anti-mouse CD16/32 (Biolegend). For the GC B cell analysis, 1×106 cells were

141

incubated with anti-mouse B220 allophycocyanin (Biolegend), anti-mouse CD95 PE

142

(eBioscience), and anti-mouse T and B cell activation marker (GL-7) FITC

143

(Biolegend). For the malaria-specific TFH cell analysis, 2×106 cells were incubated

144

with

145

(Biolegend), anti-mouse CD4 Apc/cy7 (Biolegend), anti-mouse CD11a percp/cy5.5

146

(Biolegend), anti-mouse CD49d FITC (Biolegend), anti-mouse ICOS Pe/cy7

147

(Biolegend) or anti-mouse Bcl6 PE (eBioscience) after the cells were permeabilized

148

with fixation/permeabilization agent (eBioscience). The cells were then analyzed

149

using flow cytometry.

anti-mouse

CXCR5

biotin

(Biolegend),

streptavidin-allophycocyanin

150 151 152

Statistical analysis Differences between samples were analyzed using the Graphpad Prism version 5.0. 7

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

135

153

As our data wasn’t confirmed to be normally distributed, nonparametric tests was

154

used to determine statistical significance between groups. We use Mann Whitney test

155

for the comparison of 2 groups and Kruskal-Wallis test for more than 2 groups. The p

156

values < 0.05 were considered significant.

158 159

Results

160

Absence of PD-1 greatly enhanced the protective efficacy in the ITV-immunized

161

mice

162

To determine whether PD-1-deficiency could enhance the protective efficacy in the

163

ITV-immunized mice, the parasitemia levels and survival rates of the ITV-immunized

164

WT and PD-1-deficient mice were compared after a blood-stage challenge, as

165

depicted as Fig. 1. The parasitemia curve of the PD-1-deficient mice were comparable

166

to those of WT mice, indicating that the PD-1-deficient mice had no intrinsic

167

resistance to the malaria parasite. However, compared to the non-immunized mice,

168

the growth of parasite in either ITV-immunized WT mice or PD-1-deficient mice was

169

greatly suppressed. The peak parasitemia in the immunized WT mice was

170

10.470.095%, but only 0.0170.029% in the immunized PD-1-/- mice at day 4 after

171

alive P. yoelii 17XL challenge (Fig. 1B) (p < 0.01). Parasites were cleared from all

172

immunized mice at day 8 postchallenge, and all of the mice survived (Fig. 1C). These

173

data demonstrate that PD-1-deficiency could largely augment the protective efficacy

174

of the ITV protocol. 8

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

157

175 176

Malaria parasite-specific Ab was necessary for the protective immunity of the

177

ITV-immunized mice To elucidate the mechanism of the augmented protective efficacy in the immunized

179

PD-1-/- mice, we first determined the protective immunity of the ITV-immunized mice.

180

Both Ab and CD4+ T cell responses are essential for controlling the malaria

181

blood-stage development (18). Therefore, serum from either naïve mice or

182

ITV-immunized mice was adoptively transferred to naïve mice, and the recipient mice

183

were then challenged with live P.y 17XL. The resulting parasitemia levels of the mice

184

that received naïve mice sera were comparable to those of naïve mice, and all of the

185

mice died. However, the mice that received sera from the ITV-immunized mice serum

186

cleared the parasites at day 18 post-challenge, and all of the mice survived (Fig. 2A,

187

B). These data strongly suggest that antibodies are capable of mediating protection of

188

ITV-immunized mice against malaria parasite challenge.

189

Next, the CD4+ T cells of the immunized WT mice were depleted, and the mice

190

were challenged with P.y 17XL. As shown in Fig. 2C, D, no significant difference in

191

the parasitemia or survival rate was found between the ITV-immunized mice injected

192

with control Ab and those injected with anti-CD4 Ab. Thus, in contrast to Ab, the data

193

suggest that CD4+T cells are not essential for ITV-immunized mice against the

194

blood-stage challenge.

195 196

The elevated malaria-specific Abs greatly contributed the enhanced protective 9

Downloaded from http://iai.asm.org/ on February 21, 2019 by guest

178

197

efficacy in the immunized PD-1-/- mice Because Abs are capable of mediating the protective immune response of the

199

ITV-immunized mice, the levels of malaria-specific IgG were compared between the

200

ITV-immunized WT mice and PD-1-/- mice. As shown in Fig. 3A, the levels of

201

malaria parasite-specific total IgG and isotype IgG2a in the immunized WT mice

202

were much lower than those in the immunized PD-1-/- mice (IgG, P < 0.05; IgG2a, P

203

< 0.05), although no significant difference in the IgG1 levels was found between the

204

two types of immunized mice (IgG1, P > 0.05). Thus, these data suggest that the

205

augmented protective efficacy in the immunized PD-1-/- mice was closely associated

206

with the elevated levels of malaria-specific Ab.

207

To confirm that the elevated Ab contributed to the improved protective immunity of

208

the ITV-immunized PD-1-/- mice, sera from the ITV-immunized WT mice or PD-1-/-

209

mice was adoptively transferred to the naïve mice, and the recipient mice were then

210

challenged with P.y 17XL. As a result, the appearance of parasite in the blood of the

211

mice that received the immunized PD-1-deficient mice sera was delayed by 2 days

212

compared to that of mice received the immunized WT mice sera (Fig. 3B). Although

213

all mice receiving the sera from the either immunized mice survived (Fig. 3C), the

214

parasitemia in the mice that received immunized PD-1-deficient mice sera was only

215

3.380.69%, which was much lower than that of the mice that received sera from the

216

immunized WT mice (40.865.22%) at day 8 after live P. yoelii 17XL challenge (p
Loading...

PD-1 deficiency Enhances Humoral Immunity of Malaria infection

IAI Accepted Manuscript Posted Online 2 March 2015 Infect. Immun. doi:10.1128/IAI.02621-14 Copyright © 2015, American Society for Microbiology. All Ri...

1MB Sizes 0 Downloads 0 Views

Recommend Documents

Glucose-6-phosphate Dehydrogenase Deficiency and Malaria
Abstract. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a X-chromosomally transmitted disorder of the erythrocy

Glucose-6-phosphate dehydrogenase deficiency - Malaria Journal
Abstract. Background: Extensive studies investigating the role of host genetic factors during malaria associate glucose-

Atopic dermatitis, innate immunity and infection - eScholarship
É possível encontrar dois tipos de células dendriticas na pele lesada de doentes com DA: células miéloides e células pla

Thioredoxin reductase 1 deficiency enhances selenite toxicity in
Aug 1, 2012 - Knockdown of TR1 and Trx1 in NIH 3T3 cells and of Trx1 in DT cells was carried out as described previously

Low-Calcium-Response Protein, LcrD, of - Infection and Immunity
A hydropathy analysis of the predicted proteinrevealed ... protein that has an N-terminal membrane anchor and that is im

from Lung Lavage - Infection and Immunity - American Society for
Medical Service, Veterans Administration Medical Center,1 and Division ofInfectious Diseases, University of Colorado ...

Shigella Aotus - Infection and Immunity - American Society for
Mar 3, 2014 - of Medicine and Surgery. This study was approved via Resolucion Directoral No.378 by the. 459. Directorate

(A)Historical Science - Infection and Immunity - American Society for
gard Thucydides as the first true historian and Herodotus as the “first liar” for ... chosen field of study from the

CDC - Malaria - About Malaria - Biology - Malaria Parasites
Nov 1, 2016 - Ring-form trophozoites of P. vivax in a thin blood smear. Trophozoites of P. ovale in a thin blood smear.

malaria case management - Directorate of Malaria Control
control until Pakistan joined the international partnership for Roll Back Malaria (RBM) established by the World Health