PD-1 deficiency Enhances Humoral Immunity of Malaria infection [PDF]

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 <

217

0.01). Therefore, these data strongly suggest that the elevated malaria-specific Abs

218

greatly contribute to the enhanced protective efficacy in the ITV-immunized PD-1-/10

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

198

219

mice.

220 221

The frequency and number of GC B cells significantly increased in the immunized

222

PD-1-deficient mice To further confirm the role of PD-1 signaling in the regulation of Ab production,

224

the frequencies of GC B cells in the spleen were detected at days 7, 9, 11, 14 and 21

225

after the final injection of CQ. As shown in Fig. 4, the frequency and number of GC B

226

cells in both the ITV-immunized WT mice and PD-1-/- mice gradually increased over

227

time. However, the the GC B frequency and number of the ITV-immunized PD-1-/-

228

mice was much higher than that of the ITV-immunized mice at days 14 and 21 (p <

229

0.01), but no significant difference was found either at day 7 or days 9 after the final

230

injection of CQ. These results suggest that PD-1-deficiency could promote the

231

expansion of GC B cells in the spleen of the immunized WT mice; this conclusion

232

was in agreement with the elevated level of Ab in the sera.

233 234

Plasmodium-specific TFH cells expanded in the immunized PD-1-/- mice

235

TFH cells can provide help to GC B cells for the generation of germinal centers

236

(GCs) and long-term protective humoral responses (19, 20). To test whether the

237

increased GC B cells frequency in the ITV-immunized PD-1 deficient mice was a

238

result of the expansion of TFH cells, the frequency and number of splenic TFH cells

239

were compared between the immunized WT mice and PD-1-/- mice. As described in

240

the previous study (9), we used the activation markers CD4, CXCR5, and ICOS and 11

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

223

241

the transcription factor Bcl6 to characterize splenic TFH cells. In addition, the

242

coordinated up-regulation of the integrins CD49d and CD11a on antigen-experienced

243

CD4+ T cells has also been used to identify plasmodium-specific CD4+ T cells (21).

244

Therefore,

245

CD4+CD11a+CD49d+CXCR5+Bcl6+

246

plasmodium-specific TFH cells in our study (Fig. 5A, D).

CD4+CD11a+CD49d+CXCR5+ICOS+ were

considered

as

the

247

The frequency and number of TFH cells in both the ITV-immunized WT mice and

248

PD-1-/- mice gradually reduced over time (Fig. 5B, C, E and F). The highest TFH cells

249

frequency and number were observed at day 7 after the final injection of CQ, which is

250

consistent with previous reports (22, 23), and the frequency and number were reduced

251

to the baseline level at day 21. However, the frequency and number of the

252

CD4+CD11a+CD49d+CXCR5+ICOS+

253

CD4+CD11a+CD49d+CXCR5+Bcl6+ TFH cells from the immunized PD-1-/- mice were

254

more than 3-fold greater than that of the immunized WT mice at days 7 after the

255

final injection of CQ (p < 0.01; Fig. 5). Thus, the increased malaria parasite-specific

256

TFH cells were closely associated with the expansion of GC B cells and elevated Ab in

257

the sera.

TFH

cells

or

the

258 259 260

Discussion

261

Due to the failure of malaria blood-stage subunit vaccines, whole-parasite vaccines,

262

such as ITV (7), whole-killed parasites (17) and genetically attenuated parasites (24, 12

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

cells

or

263

25), have received more attention from researchers in recent years. Understanding the

264

underlying mechanism of the whole-parasite vaccine would help us to design a more

265

effective malaria vaccine. Here, we found that the production of malaria

266

parasite-specific antibodies

267

ITV-immunized mice. Interestingly, PD-1-deficiency leads to the sterile protection of

268

the ITV-immunized mice against the malaria blood stage; this phenomenon was

269

correlated to the elevated malaria parasite-specific Ab in the serum. Additionally, the

270

elevated malaria parasite-specific Ab was closely associated with the expansion of GC

271

B cells and malaria parasite-specific TFH cells in the immunized PD-1-deficient mice.

were capable of

mediating

protection

of the

We found that the adoptive transfer of ITV-immunized mice sera could delay and

273

reduce the parasitemia after a blood-stage challenge (Fig. 2), although its effect was

274

short-term likely due to the clearance of antibodies following binding to the parasites.

275

Except for the high level of malaria-specific Ab, the possible changed antibody

276

affinity, which wasn’t tested in our study, might also contribute to the enhanced

277

protective immunity of ITV-immunized mice. However, CD4+ T cells depletion prior

278

to the challenge (day 21) did not alter the protection of the ITV-immunized mice,

279

although a great expansion of TFH was observed at early (days 7, 9, 11 and 14) after

280

the vaccination (Fig. 5). Additionally, blockade of PD-L1 and LAG-3 could promote

281

the differentiation of CD4+ TFH cells and plasmablasts in malaria infection (21).

282

These data show that TFH cells might provide the specialized help in the generation of

283

GC B cells and Ab at the early post-vaccination stage (9) but not at the late stage

284

when both GC B cells and Ab have already formed. Therefore, the protective 13

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

272

285

immunity of the ITV-immunized mice was largely dependent on the malaria

286

parasite-specific Ab, but a role for CD4+T cells to help antibody response during

287

immunization could not be completely excluded. Although ITV immunization could induce protective immunity against the blood

289

stage (6) and the liver stage (7) of the parasite, short-term parasitemia was observed

290

after challenge both in our and other studies (6). Thus, vaccinated individuals may

291

still develop clinical symptom and may be able to transmit the malaria parasite to

292

mosquitoes after malaria parasite challenge. Interestingly, we found that PD-1

293

deficiency resulted in the sterile protection of the ITV-immunized mice (Fig. 1);

294

sterile protection would prevent the development of clinical symptom and the

295

transmission of malaria by vaccinated individuals. This type of vaccine would greatly

296

contribute to the elimination of malaria worldwide.

297

Recently, evidence has shown that parasitized erythroblast could activate CD8+ T

298

cells(26). Although CD8+ T cells were found to be dispensable for the protective

299

effect of ITV-immunized mice against blood stage challenge(7), it is protective in the

300

immunized mice that survived infection with both P. yoelii XNL and, subsequently, P.

301

yoelii 17XL(27). Additionally, previous studies showed that PD-1 signaling could

302

induce CD8+ T cells anergy, not only in virus infection (28, 29), but also in chronic

303

malaria infection(30). Therefore, the contribution of CD8+ T cells to the enhanced

304

protective immunity of the ITV-immunized PD-1-deficient mice still couldn’t be

305

completely excluded.

306

Although recent studies have revealed that PD-1 signaling can also modulate the T 14

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

288

cell-dependent humoral immunity, the reports regarding the function of PD-1

308

signaling in the control of humoral immunity remain contradictory (10-14). Here, we

309

found PD-1 deficiency could significantly elevate the levels of malaria

310

parasite-specific total IgG and isotype IgG2a in the serum, and it greatly promoted the

311

expansion of both GC B cells and TFH cells in the ITV-immunized mice. Serum

312

adoptive transfer assays further confirmed the negative role of PD-1 signaling in the

313

control of humoral immunity. This is consistent with a negative regulatory role of

314

PD-1 signaling in the regulation of TFH in chronic malaria infections (21).

315

PD-1 has two known ligands, PD-L1 and PD-L2. PD-L1 is expressed on a wider

316

range of cells than PD-L2, but both of them can be expressed on GC B cells and

317

dendritic cells (31). Although PD-1/PD-L1 or PD-1/PD-L2 signaling has been

318

reported to modulate TFH cells, GC B cells and Ab (11, 12), the ligand that is involved

319

in the humoral immunity of ITV-immunized mice remains to be determined. Recently,

320

follicular regulatory T cells (TFR cells) that suppress the germinal center reaction were

321

identified (32, 33). Because FoxP3 is the only marker that distinguishes TFR from TFH,

322

it seems likely that previously identified ‘TFH cells’ with markers of ICOS, CXCR5

323

and PD-1 could be mixtures of stimulatory TFH cells and inhibitory TFR cells.

324

Therefore, the effect of PD-1-deficiency on TFH and TFR cells in the ITV-immunized

325

mice warrants further investigation.

326

In conclusion, we demonstrate that malaria parasite-specific Ab are capable of

327

mediating the protective immunity of the ITV-immunized mice. Interestingly, PD-1

328

deficiency could confer sterile protective immunity to the ITV-immunized mice; this 15

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

307

is an important step in the worldwide elimination of malaria. We also provide

330

evidence that PD-1 signaling could greatly enhance the malaria specific B cells

331

response and the expansion of TFH cells, further supporting the negative role of PD-1

332

signaling in the modulation of humoral immunity. Thus, our findings have

333

implications not only for the rational design of an effective blood-stage vaccine

334

against malaria parasites through the induction of a robust B cells response but also

335

further our understanding of the regulatory role of PD-1 signaling in the humoral

336

immune response.

337 338 339

Acknowledgments

340

This work was supported by the National Science Foundation of China (81271859),

341

the Natural Science Foundation of PLA (CWS12J093), and Major Project of PLA

342

(BWS11J041).

343

We also thank W. Peters and B.L. Robinson from the Malaria Research and Reference

344

Reagent Resource Center for providing P. yoelii 17XL.

345 346

References

347 348 349 350 351 352 353 354

1.

WHO. 2013. World malaria report 2013. World Health Organization, Geneva, Switzerland:http://www.who.int/malaria/publications/world_malaria_report_2013/en/.

2.

Delany I, Rappuoli R, De Gregorio E. 2014. Vaccines for the 21st century. EMBO Mol Med 6:708-720.

3.

Genton B, Reed ZH. 2007. Asexual blood-stage malaria vaccine development: facing the challenges. Curr Opin Infect Dis 20:467-475.

4.

Cowman AF, Crabb BS. 2006. Invasion of red blood cells by malaria parasites. Cell 124:755-766. 16

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

329

5.

Mu J, Awadalla P, Duan J, McGee KM, Keebler J, Seydel K, McVean GA, Su XZ. 2007. Genome-wide variation and identification of vaccine targets in the Plasmodium falciparum genome. Nat Genet 39:126-130.

6.

Elliott SR, Kuns RD, Good MF. 2005. Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria. Infect Immun 73:2478-2485.

7.

Belnoue E, Voza T, Costa FT, Gruner AC, Mauduit M, Rosa DS, Depinay N, Kayibanda M, Vigario AM, Mazier D, Snounou G, Sinnis P, Renia L. 2008. Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage. J Immunol 181:8552-8558.

8.

Pombo DJ, Lawrence G, Hirunpetcharat C, Rzepczyk C, Bryden M, Cloonan N, Anderson K, Mahakunkijcharoen Y, Martin LB, Wilson D, Elliott S, Eisen DP, Weinberg JB, Saul A, Good MF. 2002. Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 360:610-617.

9.

Crotty S. 2011. Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29:621-663.

10.

Velu V, Titanji K, Zhu B, Husain S, Pladevega A, Lai L, Vanderford TH, Chennareddi L, Silvestri G, Freeman GJ, Ahmed R, Amara RR. 2009. Enhancing SIV-specific immunity in vivo by PD-1 blockade. Nature 458:206-210.

11.

Hams E, McCarron MJ, Amu S, Yagita H, Azuma M, Chen L, Fallon PG. 2011. Blockade of B7-H1 (programmed death ligand 1) enhances humoral immunity by positively regulating the generation of T follicular helper cells. J Immunol 186:5648-5655.

12.

Good-Jacobson KL, Szumilas CG, Chen L, Sharpe AH, Tomayko MM, Shlomchik MJ. 2010. PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells. Nat Immunol 11:535-542.

13.

Hamel KM, Cao Y, Wang Y, Rodeghero R, Kobezda T, Chen L, Finnegan A. 2010. B7-H1 expression on non-B and non-T cells promotes distinct effects on T- and B-cell responses in autoimmune arthritis. Eur J Immunol 40:3117-3127.

14.

Kawamoto S, Tran TH, Maruya M, Suzuki K, Doi Y, Tsutsui Y, Kato LM, Fagarasan S. 2012. The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut. Science 336:485-489.

15.

Liu T, Xu G, Guo B, Fu Y, Qiu Y, Ding Y, Zheng H, Fu X, Wu Y, Xu W. 2013. An essential role for C5aR signaling in the optimal induction of a malaria-specific CD4+ T cell response by a whole-killed blood-stage vaccine. J Immunol 191:178-186.

16.

Abes R, Gelize E, Fridman WH, Teillaud JL. 2010. Long-lasting antitumor protection by anti-CD20 antibody through cellular immune response. Blood 116:926-934.

17.

Pinzon-Charry A, McPhun V, Kienzle V, Hirunpetcharat C, Engwerda C, McCarthy J, Good MF. 2010. Low doses of killed parasite in CpG elicit vigorous CD4+ T cell responses against blood-stage malaria in mice. J Clin Invest 120:2967-2978.

18.

Good MF, Xu H, Wykes M, Engwerda CR. 2005. Development and regulation of cell-mediated immune responses to the blood stages of malaria: implications for vaccine research. Annu Rev Immunol 23:69-99.

19.

Gowthaman U, Chodisetti SB, Agrewala JN. 2010. T cell help to B cells in germinal centers: putting the jigsaw together. Int Rev Immunol 29:403-420.

20.

Nutt SL, Tarlinton DM. 2011. Germinal center B and follicular helper T cells: siblings, 17

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

355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398

cousins or just good friends? Nat Immunol 12:472-477. 21.

Butler NS, Moebius J, Pewe LL, Traore B, Doumbo OK, Tygrett LT, Waldschmidt TJ, Crompton PD, Harty JT. 2011. Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol 13:188-195.

22.

Seung Goo Kang W-HL, Peiwen Lu, Shepherd, Daniel Fremgen, Teijaro

Hyun Yong Jin,

Eric Verdin,

Hyung W Lim,

Michael B A Oldstone, Hai Qi,

Jovan John R

& Changchun Xiao. 2013. MicroRNAs of the miR-17~92 family are critical

regulators of TFH differentiation. NATURE IMMUNOLOGY 14:849–857. 23.

Linterman MA, Beaton L, Yu D, Ramiscal RR, Srivastava M, Hogan JJ, Verma NK, Smyth MJ, Rigby RJ, Vinuesa CG. 2010. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J Exp Med 207:353-363.

24.

Ting LM, Gissot M, Coppi A, Sinnis P, Kim K. 2008. Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity. Nat Med 14:954-958.

25.

Aly AS, Downie MJ, Mamoun CB, Kappe SH. 2010. Subpatent infection with nucleoside transporter 1-deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice. Cell Microbiol 12:930-938.

26.

Imai T, Ishida H, Suzue K, Hirai M, Taniguchi T, Okada H, Suzuki T, Shimokawa C, Hisaeda H. 2013. CD8(+) T cell activation by murine erythroblasts infected with malaria parasites. Sci Rep 3:1572.

27.

Imai T, Shen J, Chou B, Duan X, Tu L, Tetsutani K, Moriya C, Ishida H, Hamano S, Shimokawa C, Hisaeda H, Himeno K. 2010. Involvement of CD8+ T cells in protective immunity against murine blood-stage infection with Plasmodium yoelii 17XL strain. Eur J Immunol 40:1053-1061.

28.

Maier H, Isogawa M, Freeman GJ, Chisari FV. 2007. PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver. J Immunol 178:2714-2720.

29.

Lukens JR, Cruise MW, Lassen MG, Hahn YS. 2008. Blockade of PD-1/B7-H1 interaction restores effector CD8+ T cell responses in a hepatitis C virus core murine model. J Immunol 180:4875-4884.

30.

Horne-Debets JM, Faleiro R, Karunarathne DS, Liu XQ, Lineburg KE, Poh CM, Grotenbreg GM, Hill GR, Macdonald KP, Good MF, Renia L, Ahmed R, Sharpe AH, Wykes MN. 2013. PD-1 Dependent Exhaustion of CD8(+) T Cells Drives Chronic Malaria. Cell Rep 5:1204-1213.

31.

Keir ME, Butte MJ, Freeman GJ, Sharpe AH. 2008. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677-704.

32.

Chung Y, Tanaka S, Chu F, Nurieva RI, Martinez GJ, Rawal S, Wang YH, Lim H, Reynolds JM, Zhou XH, Fan HM, Liu ZM, Neelapu SS, Dong C. 2011. Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med 17:983-988.

33.

Linterman MA, Pierson W, Lee SK, Kallies A, Kawamoto S, Rayner TF, Srivastava M, Divekar DP, Beaton L, Hogan JJ, Fagarasan S, Liston A, Smith KG, Vinuesa CG. 2011. Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med 17:975-982.

18

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

399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442

Figure Legends:

444

Figure 1: The protective efficacy against blood-stage challenge was markedly

445

enhanced in the ITV-immunized PD-1-/- mice. A, The procedure for the ITV

446

immunization and challenge. B and C, Naïve or immunized WT (n = 5) and PD-1-/- (n

447

= 5) mice were challenged i.p. with P. yoelii 17XL pRBC at day 21 after the last CQ

448

injection. The parasitemia (B) and survival rate (C) were recorded. The results are

449

representative of three independent experiments. The data are presented as the mean ±

450

SD; **p < 0.01.

451 452

Figure 2: The protective immunity of the ITV-immunized WT mice. A and B, Sera

453

from the naïve mice or ITV-immunized WT mice were adoptively transferred into

454

each naïve mouse (n = 3) at days -1, 0 and 1. All mice were challenged with P. yoelii

455

17XL on day 0, and the parasitemia (A) and survival rate (B) were determined. C and

456

D, On day -1 and 1 before the challenge, immunized WT mice (n = 3) were injected

457

with anti-CD4 or control IgG. Then, all mice were challenged with P. yoelii 17XL on

458

day 0, and the parasitemia (C) and survival rate (D) were monitored. All experiments

459

were performed twice. The data are presented as the mean ± SD.

460 461

Figure 3: The elevated malaria-specific Ab contributed to the enhanced protective

462

efficacy of the ITV-immunized PD-1-/- mice. A, Three weeks after the final

463

immunization, the levels of total IgG, IgG1, and IgG2a in the sera of both immunized

464

WT (n = 5) and PD-1-/- mice (n = 5) were detected by ELISA. Sera from 19

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

443

PBS-immunized mice served as the negative control, and HIS served as the positive

466

control. The data are presented as the lg of Ab titer. B and C, Sera from the naïve mice,

467

ITV-immunized WT mice or PD-1-/- mice were adoptively transferred into each naïve

468

mouse (n = 3) at day -1, 0 and 1, and all mice were then subsequently challenged with

469

P. yoelii 17XL. The parasitemia (B) and survival rate (C) were determined. **p <

470

0.01.

471 472

Figure 4: The frequency and number of GC B cells in the spleens of the

473

ITV-immunized WT and PD-1-/- mice. Splenocytes were isolated from the immunized

474

WT and PD-1-/- mice at the indicated time after the final immunization, and both the

475

frequency and number of GC B cells was analyzed by FACS. A, Representative FACS

476

analysis of B220+GL-7+CD95+ GC B cells at days 7, 14 and 21. B and C, Statistical

477

analysis of the frequency (B) and number (C) of GC B cells from the ITV-immunized

478

WT and PD-1-/- mice at days 7, 9, 11, 14 and 21. Three individual experiments were

479

performed. The data are presented as the mean ± SD; ns, no significance; *p < 0.05;

480

**p < 0.01.

481 482

Figure 5: The frequency and number of malaria-specific TFH cells from the

483

ITV-immunized WT and PD-1-/- mice. Splenocytes were isolated from the immunized

484

WT (n=5) and PD-1-/- mice (n = 5) at the indicated day after the final immunization,

485

and the TFH cells were analyzed by FACS. A, Representative FACS analysis of

486

CD4+CD11a+CD49+CXCR5+ICOS+ malaria-specific TFH cells; B and C, Statistical 20

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

465

analysis of the frequency (B) and number (C) of CD4+CD11a+CD49+CXCR5+ICOS+

488

cells in the immunized WT and PD-1-/- mice. D, Representative FACS analysis of

489

CD4+CD11a+CD49+CXCR5+Bcl6+ malaria-specific TFH cells at days 7, 14 and 21. E

490

and F, Statistical analysis of the frequency (E) and number (F) TFH cells

491

(CD4+CD11a+CD49+CXCR5+Bcl6+) from the immunized WT and PD-1-/- mice at

492

days 7, 9, 11, 14 and 21. Three experiments were performed. The data are presented

493

as the mean ±SD; *p< 0.05; **p< 0.01.

21

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

487

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

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

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

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

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