1

PD-1 deficiency Enhances Humoral Immunity of Malaria 1

infection-treated vaccine 2

3

Taiping Liu1, Xiao Lu

1, Chenghao Zhao

1, Xiaolan Fu

2, Tingting Zhao

2#, Wenyue Xu

1# 4

1Department of Pathogenic Biology, Third Military Medical University, Chongqing, P. 5

R. China. 6

2Institute of Immunology of PLA, Third Military Medical University, Chongqing, P. R. 7

China. 8

9

Running title: PD-1 deficiency and humoral immunity 10

These authors declare that there is no conflict of interest. 11

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: xuwenyue@gmail.com; or Tingting Zhao, Institute of Immunology

of PLA, Third Military Medical University, Chongqing, P.R. China. E-mail:

772108644@qq.com.

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

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Abstract 21

Malaria infection-treatment vaccine (ITV) is a promising strategy to induce 22

homologous and heterologous protective immunity against the blood stage of the 23

parasite. However, the underlying mechanism of protection remains largely unknown. 24

Here, we found that a malaria-specific Ab could mediate the protective immunity of 25

the ITV-immunized mice. Interestingly, PD-1 deficiency greatly elevated the levels of 26

both malaria-specific total IgG and subclass IgG2a and enhanced the protective 27

efficacy of ITV-immunized mice against the blood-stage challenge. A serum adoptive 28

transfer assay demonstrated that the increased Ab level contributed to the enhanced 29

protective efficacy of the immunized PD-1-deficient mice. Further study showed that 30

PD-1 deficiency could also promote the expansion of germinal center (GC) B cells 31

and malaria parasite-specific TFH cells in the spleens of the ITV-immunized mice. 32

These results implicate that PD-1 deficiency improves the protective efficacy of 33

ITV-immunized mice by promoting the generation of malaria parasite-specific Ab and 34

the expansion of GC B cells. The results of this study provide us with new evidence to 35

support the negative function PD-1 on humoral immunity and will guide the design of 36

a more effective malaria vaccine. 37

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Introduction 43

Although malaria control programs have led to an extensive reduction in malaria 44

incidence and mortality, it remains one of the most threatening diseases worldwide. It 45

is estimated that 207 million cases and 627,000 malaria deaths occurred in 2012 (1). 46

A vaccine is regarded as the most cost-effective strategy to prevent malaria 47

infection (2). Most malaria subunit blood-stage vaccines have been designed to induce 48

antibodies (Ab) against a variety of surface proteins on the merozoite to block the 49

invasion of red blood cells (3). However, the invasion of the merozoites into red blood 50

cells is controlled by multiple redundant proteins (4), and Ab against one or two 51

merozoite surface proteins are unable to effectively prevent the infection of red blood 52

cells with the malaria parasite (4). Furthermore, most merozoite surface proteins 53

exhibit antigenic polymorphism under selective pressure (5). To date, there is no 54

malaria subunit vaccine available worldwide. 55

In contrast to the subunit malaria vaccine, the malaria 56

infection-treatment-vaccination (ITV), which involves infection with live malaria 57

parasites under curative anti-malarial drug coverage, has been reported to induce 58

antibodies specific for the merozoite surface antigens conserved between 59

heterologous strains but not for the variant surface antigens (6). ITV induces strong 60

protective immunity against the blood-stage of the parasite in animals (7) and humans 61

(8). Interestingly, ITV can also confer cross-protection against the liver stage of 62

malaria by inducing cellular immune responses (7). However, the underlying 63

mechanism of protective immunity induced by ITV is still largely unknown. 64

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Follicular helper CD4 T(TFH) cells are characterized by the high expression of 65

chemokine receptor CXCR5, programmed death 1 (PD-1), lineage-specific 66

transcription regulator Bcl6, SAP (SH2D1A), IL-21, and ICOS and are recognized as 67

specialized providers of cognate B cell help (9). Of these characteristic molecules, 68

PD-1 has been reported to provide modulatory signals to GC TFH cells, but its 69

function in the modulation of humoral immunity remains unresolved. Some evidence 70

has shown that the blockade of PD-L1 or PD-1 reinforces TFH cell expansion, 71

increases the number of GC B cells and plasmablasts and enhances antigen-specific 72

Ab responses (10, 11). However, attenuated humoral immune responses also have 73

been observed after blockade of PD-1 signaling (12-14). Therefore, the exact role of 74

PD-1 signaling in the protective immunity of the ITV-immunized mice remains 75

unclear. 76

In this study, we found that PD-1-deficiency greatly improved the protective 77

efficacy of ITV-immunized mice against a malaria blood stage challenge. This 78

phenomenon was attributed to the elevated malaria parasite-specific Ab in the 79

immunized PD-1-deficient mice. Additionally, we also observed increased GC B cells 80

and the expansion of TFH cells in the immunized PD-1-deficient mice. Thus, our data 81

further confirmed the negative effect of PD-1 signaling on humoral immunity and 82

shed new light on the design of effective malaria vaccine. 83

84

85

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Materials and Methods 87

Mice and Plasmodium parasites 88

PD-1-/-

mice (BALB/c background) were obtained from the Jackson Laboratory 89

(Bar Harbor, ME). Specific pathogen-free BALB/c mice, at 6-8 week of age, were 90

purchased from the Beijing Animal Institute. All animal protocols were reviewed and 91

approved by the Animal Ethics Committee of the Third Military Medical University 92

Institute of Medical Research. The lethal strain Plasmodium yoelii 17XL was obtained 93

from MR4 (Malaria Research and Reference Reagent Resource Center, Manassas, 94

Virginia) and maintained by i.p. passages in mice. 95

96

Immunization and challenge 97

The immunization schedule was performed as previously described (7) with minor 98

modifications. Briefly, mice were i.v. injected with 1×106

P. yoelii 17XL live iRBC 99

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

mg/ml chloroquine (CQ, Sigma-Aldrich) diluted in saline daily for 15 days starting 101

from the day of iRBC injection. The mice were maintained for 21 days after the last 102

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

103

Py-iRBC. 104

105

Adoptive serum transfer assay and CD4+T cell depletion 106

For serum transfer, naïve BALB/c mice were i.v. injected on days -1, 0 and 1 with 107

0.2 ml naïve mice serum, ITV-immunized WT or PD-1-/-

mice serum collected 21 days 108

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after the last CQ injection, as described previously (15). The mice were challenged 109

with 2.5×104

Py-iRBC on day 0, and parasitemia and the survival rate were 110

determined. For CD4 depletion studies, an anti-CD4-depleting mAb (GK1.5 clone, 111

200 µg per mouse in 200 µl of PBS; BioXcell) or control Ab was i.p. injected on days 112

-1 and 1 (20 and 22 days after the last CQ injection) according to a previously 113

described protocol(16). CD4+

T cell depletion was verified by staining blood samples 114

with anti-CD4 (clone RM4-5, eBioscience). The mice were then challenged with 115

1×106

Py-iRBC on day 0. 116

117

Detection of malaria-specific IgG in serum 118

The serum was collected from the naïve mice, ITV-immunized WT and PD-1-/- 119

mice at 21 days after the last CQ injection. Hyperimmune sera (HIS) were collected 120

from the ITV-immunized WT mice that had recovered from the P. yoelii 17XL 121

infection. The malaria-specific total IgG, IgG1 and IgG2a in the serum were detected 122

as previously described (15, 17). Briefly, P. yoelii 17XL-infected mouse blood was 123

collected, lysed with 0.01% saponin (Sigma-Aldrich) at 37°C for 20 min, and 124

sonicated in PBS. NuncMaxiSorp Immunoplates (NalgeNunc) were coated with 125

parasite Ag at a concentration of 5-10 μg/ml overnight at 4°C and co-incubated with 126

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

mice. 127

Biotin-conjugated anti-mouse IgG1 and IgG2a (Biolegend) were added to the plates 128

to detect IgG1 and IgG2a. After washing with wash buffer, the plates were incubated 129

with HRP-conjugated anti-mouse IgG or HRP-conjugated streptavidin (Biolegend), 130

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and 3,3’,5,5’-tetramethylbenzidine was added (Biolegend). The absorbance at wave 131

length of 450 nm was read using a spectrophotometer. 132

133

Flow cytometry analysis of GC B and Malaria-specific TFH cells 134

Both GC B and malaria-specific TFH cells from the naïve mice, ITV-immunized 135

WT and PD-1-/- mice were analyzed at 7, 9, 11, 14 and 21 days (at 21, 23, 25, 28 and 136

35 days after the start of CQ treatment), respectively, after the last CQ injection. In 137

brief, single-cell suspensions of splenocytes were prepared and washed in flow 138

cytometry buffer (PBS with 2% FCS and 0.05% sodium azide) followed by blocking 139

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

cells were 140

incubated with anti-mouse B220 allophycocyanin (Biolegend), anti-mouse CD95 PE 141

(eBioscience), and anti-mouse T and B cell activation marker (GL-7) FITC 142

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

cells were incubated 143

with anti-mouse CXCR5 biotin (Biolegend), streptavidin-allophycocyanin 144

(Biolegend), anti-mouse CD4 Apc/cy7 (Biolegend), anti-mouse CD11a percp/cy5.5 145

(Biolegend), anti-mouse CD49d FITC (Biolegend), anti-mouse ICOS Pe/cy7 146

(Biolegend) or anti-mouse Bcl6 PE (eBioscience) after the cells were permeabilized 147

with fixation/permeabilization agent (eBioscience). The cells were then analyzed 148

using flow cytometry. 149

150

Statistical analysis 151

Differences between samples were analyzed using the Graphpad Prism version 5.0. 152

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As our data wasn’t confirmed to be normally distributed, nonparametric tests was 153

used to determine statistical significance between groups. We use Mann Whitney test 154

for the comparison of 2 groups and Kruskal-Wallis test for more than 2 groups. The p 155

values < 0.05 were considered significant. 156

157

158

Results 159

Absence of PD-1 greatly enhanced the protective efficacy in the ITV-immunized 160

mice 161

To determine whether PD-1-deficiency could enhance the protective efficacy in the 162

ITV-immunized mice, the parasitemia levels and survival rates of the ITV-immunized 163

WT and PD-1-deficient mice were compared after a blood-stage challenge, as 164

depicted as Fig. 1. The parasitemia curve of the PD-1-deficient mice were comparable 165

to those of WT mice, indicating that the PD-1-deficient mice had no intrinsic 166

resistance to the malaria parasite. However, compared to the non-immunized mice, 167

the growth of parasite in either ITV-immunized WT mice or PD-1-deficient mice was 168

greatly suppressed. The peak parasitemia in the immunized WT mice was 169

10.470.095%, but only 0.0170.029% in the immunized PD-1-/-

mice at day 4 after 170

alive P. yoelii 17XL challenge (Fig. 1B) (p < 0.01). Parasites were cleared from all 171

immunized mice at day 8 postchallenge, and all of the mice survived (Fig. 1C). These 172

data demonstrate that PD-1-deficiency could largely augment the protective efficacy 173

of the ITV protocol. 174

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175

Malaria parasite-specific Ab was necessary for the protective immunity of the 176

ITV-immunized mice 177

To elucidate the mechanism of the augmented protective efficacy in the immunized 178

PD-1-/-

mice, we first determined the protective immunity of the ITV-immunized mice. 179

Both Ab and CD4+ T cell responses are essential for controlling the malaria 180

blood-stage development (18). Therefore, serum from either naïve mice or 181

ITV-immunized mice was adoptively transferred to naïve mice, and the recipient mice 182

were then challenged with live P.y 17XL. The resulting parasitemia levels of the mice 183

that received naïve mice sera were comparable to those of naïve mice, and all of the 184

mice died. However, the mice that received sera from the ITV-immunized mice serum 185

cleared the parasites at day 18 post-challenge, and all of the mice survived (Fig. 2A, 186

B). These data strongly suggest that antibodies are capable of mediating protection of 187

ITV-immunized mice against malaria parasite challenge. 188

Next, the CD4+ T cells of the immunized WT mice were depleted, and the mice 189

were challenged with P.y 17XL. As shown in Fig. 2C, D, no significant difference in 190

the parasitemia or survival rate was found between the ITV-immunized mice injected 191

with control Ab and those injected with anti-CD4 Ab. Thus, in contrast to Ab, the data 192

suggest that CD4+T cells are not essential for ITV-immunized mice against the 193

blood-stage challenge. 194

195

The elevated malaria-specific Abs greatly contributed the enhanced protective 196

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efficacy in the immunized PD-1-/-

mice 197

Because Abs are capable of mediating the protective immune response of the 198

ITV-immunized mice, the levels of malaria-specific IgG were compared between the 199

ITV-immunized WT mice and PD-1-/-

mice. As shown in Fig. 3A, the levels of 200

malaria parasite-specific total IgG and isotype IgG2a in the immunized WT mice 201

were much lower than those in the immunized PD-1-/-

mice (IgG, P < 0.05; IgG2a, P 202

< 0.05), although no significant difference in the IgG1 levels was found between the 203

two types of immunized mice (IgG1, P > 0.05). Thus, these data suggest that the 204

augmented protective efficacy in the immunized PD-1-/-

mice was closely associated 205

with the elevated levels of malaria-specific Ab. 206

To confirm that the elevated Ab contributed to the improved protective immunity of 207

the ITV-immunized PD-1-/-

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

208

mice was adoptively transferred to the naïve mice, and the recipient mice were then 209

challenged with P.y 17XL. As a result, the appearance of parasite in the blood of the 210

mice that received the immunized PD-1-deficient mice sera was delayed by 2 days 211

compared to that of mice received the immunized WT mice sera (Fig. 3B). Although 212

all mice receiving the sera from the either immunized mice survived (Fig. 3C), the 213

parasitemia in the mice that received immunized PD-1-deficient mice sera was only 214

3.380.69%, which was much lower than that of the mice that received sera from the 215

immunized WT mice (40.865.22%) at day 8 after live P. yoelii 17XL challenge (p < 216

0.01). Therefore, these data strongly suggest that the elevated malaria-specific Abs 217

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

218

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mice. 219

220

The frequency and number of GC B cells significantly increased in the immunized 221

PD-1-deficient mice 222

To further confirm the role of PD-1 signaling in the regulation of Ab production, 223

the frequencies of GC B cells in the spleen were detected at days 7, 9, 11, 14 and 21 224

after the final injection of CQ. As shown in Fig. 4, the frequency and number of GC B 225

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

mice gradually increased over 226

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

227

mice was much higher than that of the ITV-immunized mice at days 14 and 21 (p < 228

0.01), but no significant difference was found either at day 7 or days 9 after the final 229

injection of CQ. These results suggest that PD-1-deficiency could promote the 230

expansion of GC B cells in the spleen of the immunized WT mice; this conclusion 231

was in agreement with the elevated level of Ab in the sera. 232

233

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

mice 234

TFH cells can provide help to GC B cells for the generation of germinal centers 235

(GCs) and long-term protective humoral responses (19, 20). To test whether the 236

increased GC B cells frequency in the ITV-immunized PD-1 deficient mice was a 237

result of the expansion of TFH cells, the frequency and number of splenic TFH cells 238

were compared between the immunized WT mice and PD-1-/-

mice. As described in 239

the previous study (9), we used the activation markers CD4, CXCR5, and ICOS and 240

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the transcription factor Bcl6 to characterize splenic TFH cells. In addition, the 241

coordinated up-regulation of the integrins CD49d and CD11a on antigen-experienced 242

CD4+ T cells has also been used to identify plasmodium-specific CD4

+ T cells (21). 243

Therefore, CD4+CD11a

+CD49d

+CXCR5

+ICOS

+ or 244

CD4+CD11a

+CD49d

+CXCR5

+Bcl6

+ cells were considered as the 245

plasmodium-specific TFH cells in our study (Fig. 5A, D). 246

The frequency and number of TFH cells in both the ITV-immunized WT mice and 247

PD-1-/-

mice gradually reduced over time (Fig. 5B, C, E and F). The highest TFH cells 248

frequency and number were observed at day 7 after the final injection of CQ, which is 249

consistent with previous reports (22, 23), and the frequency and number were reduced 250

to the baseline level at day 21. However, the frequency and number of the 251

CD4+CD11a

+CD49d

+CXCR5

+ICOS

+ TFH cells or the 252

CD4+CD11a

+CD49d

+CXCR5

+Bcl6

+ TFH cells from the immunized PD-1

-/- mice were 253

more than 3-fold greater than that of the immunized WT mice at days 7 after the 254

final injection of CQ (p < 0.01; Fig. 5). Thus, the increased malaria parasite-specific 255

TFH cells were closely associated with the expansion of GC B cells and elevated Ab in 256

the sera. 257

258

259

Discussion 260

Due to the failure of malaria blood-stage subunit vaccines, whole-parasite vaccines, 261

such as ITV (7), whole-killed parasites (17) and genetically attenuated parasites (24, 262

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25), have received more attention from researchers in recent years. Understanding the 263

underlying mechanism of the whole-parasite vaccine would help us to design a more 264

effective malaria vaccine. Here, we found that the production of malaria 265

parasite-specific antibodies were capable of mediating protection of the 266

ITV-immunized mice. Interestingly, PD-1-deficiency leads to the sterile protection of 267

the ITV-immunized mice against the malaria blood stage; this phenomenon was 268

correlated to the elevated malaria parasite-specific Ab in the serum. Additionally, the 269

elevated malaria parasite-specific Ab was closely associated with the expansion of GC 270

B cells and malaria parasite-specific TFH cells in the immunized PD-1-deficient mice. 271

We found that the adoptive transfer of ITV-immunized mice sera could delay and 272

reduce the parasitemia after a blood-stage challenge (Fig. 2), although its effect was 273

short-term likely due to the clearance of antibodies following binding to the parasites. 274

Except for the high level of malaria-specific Ab, the possible changed antibody 275

affinity, which wasn’t tested in our study, might also contribute to the enhanced 276

protective immunity of ITV-immunized mice. However, CD4+

T cells depletion prior 277

to the challenge (day 21) did not alter the protection of the ITV-immunized mice, 278

although a great expansion of TFH was observed at early (days 7, 9, 11 and 14) after 279

the vaccination (Fig. 5). Additionally, blockade of PD-L1 and LAG-3 could promote 280

the differentiation of CD4+ TFH cells and plasmablasts in malaria infection (21). 281

These data show that TFH cells might provide the specialized help in the generation of 282

GC B cells and Ab at the early post-vaccination stage (9) but not at the late stage 283

when both GC B cells and Ab have already formed. Therefore, the protective 284

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immunity of the ITV-immunized mice was largely dependent on the malaria 285

parasite-specific Ab, but a role for CD4+T cells to help antibody response during 286

immunization could not be completely excluded. 287

Although ITV immunization could induce protective immunity against the blood 288

stage (6) and the liver stage (7) of the parasite, short-term parasitemia was observed 289

after challenge both in our and other studies (6). Thus, vaccinated individuals may 290

still develop clinical symptom and may be able to transmit the malaria parasite to 291

mosquitoes after malaria parasite challenge. Interestingly, we found that PD-1 292

deficiency resulted in the sterile protection of the ITV-immunized mice (Fig. 1); 293

sterile protection would prevent the development of clinical symptom and the 294

transmission of malaria by vaccinated individuals. This type of vaccine would greatly 295

contribute to the elimination of malaria worldwide. 296

Recently, evidence has shown that parasitized erythroblast could activate CD8+ T 297

cells(26). Although CD8+ T cells were found to be dispensable for the protective 298

effect of ITV-immunized mice against blood stage challenge(7), it is protective in the 299

immunized mice that survived infection with both P. yoelii XNL and, subsequently, P. 300

yoelii 17XL(27). Additionally, previous studies showed that PD-1 signaling could 301

induce CD8+ T cells anergy, not only in virus infection (28, 29), but also in chronic 302

malaria infection(30). Therefore, the contribution of CD8+ T cells to the enhanced 303

protective immunity of the ITV-immunized PD-1-deficient mice still couldn’t be 304

completely excluded. 305

Although recent studies have revealed that PD-1 signaling can also modulate the T 306

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cell-dependent humoral immunity, the reports regarding the function of PD-1 307

signaling in the control of humoral immunity remain contradictory (10-14). Here, we 308

found PD-1 deficiency could significantly elevate the levels of malaria 309

parasite-specific total IgG and isotype IgG2a in the serum, and it greatly promoted the 310

expansion of both GC B cells and TFH cells in the ITV-immunized mice. Serum 311

adoptive transfer assays further confirmed the negative role of PD-1 signaling in the 312

control of humoral immunity. This is consistent with a negative regulatory role of 313

PD-1 signaling in the regulation of TFH in chronic malaria infections (21). 314

PD-1 has two known ligands, PD-L1 and PD-L2. PD-L1 is expressed on a wider 315

range of cells than PD-L2, but both of them can be expressed on GC B cells and 316

dendritic cells (31). Although PD-1/PD-L1 or PD-1/PD-L2 signaling has been 317

reported to modulate TFH cells, GC B cells and Ab (11, 12), the ligand that is involved 318

in the humoral immunity of ITV-immunized mice remains to be determined. Recently, 319

follicular regulatory T cells (TFR cells) that suppress the germinal center reaction were 320

identified (32, 33). Because FoxP3 is the only marker that distinguishes TFR from TFH, 321

it seems likely that previously identified ‘TFH cells’ with markers of ICOS, CXCR5 322

and PD-1 could be mixtures of stimulatory TFH cells and inhibitory TFR cells. 323

Therefore, the effect of PD-1-deficiency on TFH and TFR cells in the ITV-immunized 324

mice warrants further investigation. 325

In conclusion, we demonstrate that malaria parasite-specific Ab are capable of 326

mediating the protective immunity of the ITV-immunized mice. Interestingly, PD-1 327

deficiency could confer sterile protective immunity to the ITV-immunized mice; this 328

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is an important step in the worldwide elimination of malaria. We also provide 329

evidence that PD-1 signaling could greatly enhance the malaria specific B cells 330

response and the expansion of TFH cells, further supporting the negative role of PD-1 331

signaling in the modulation of humoral immunity. Thus, our findings have 332

implications not only for the rational design of an effective blood-stage vaccine 333

against malaria parasites through the induction of a robust B cells response but also 334

further our understanding of the regulatory role of PD-1 signaling in the humoral 335

immune response. 336

337

338

Acknowledgments 339

This work was supported by the National Science Foundation of China (81271859), 340

the Natural Science Foundation of PLA (CWS12J093), and Major Project of PLA 341

(BWS11J041). 342

We also thank W. Peters and B.L. Robinson from the Malaria Research and Reference 343

Reagent Resource Center for providing P. yoelii 17XL. 344

345

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Figure Legends: 443

Figure 1: The protective efficacy against blood-stage challenge was markedly 444

enhanced in the ITV-immunized PD-1-/-

mice. A, The procedure for the ITV 445

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

(n 446

= 5) mice were challenged i.p. with P. yoelii 17XL pRBC at day 21 after the last CQ 447

injection. The parasitemia (B) and survival rate (C) were recorded. The results are 448

representative of three independent experiments. The data are presented as the mean ± 449

SD; **p < 0.01. 450

451

Figure 2: The protective immunity of the ITV-immunized WT mice. A and B, Sera 452

from the naïve mice or ITV-immunized WT mice were adoptively transferred into 453

each naïve mouse (n = 3) at days -1, 0 and 1. All mice were challenged with P. yoelii 454

17XL on day 0, and the parasitemia (A) and survival rate (B) were determined. C and 455

D, On day -1 and 1 before the challenge, immunized WT mice (n = 3) were injected 456

with anti-CD4 or control IgG. Then, all mice were challenged with P. yoelii 17XL on 457

day 0, and the parasitemia (C) and survival rate (D) were monitored. All experiments 458

were performed twice. The data are presented as the mean ± SD. 459

460

Figure 3: The elevated malaria-specific Ab contributed to the enhanced protective 461

efficacy of the ITV-immunized PD-1-/-

mice. A, Three weeks after the final 462

immunization, the levels of total IgG, IgG1, and IgG2a in the sera of both immunized 463

WT (n = 5) and PD-1-/-

mice (n = 5) were detected by ELISA. Sera from 464

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PBS-immunized mice served as the negative control, and HIS served as the positive 465

control. The data are presented as the lg of Ab titer. B and C, Sera from the naïve mice, 466

ITV-immunized WT mice or PD-1-/- mice were adoptively transferred into each naïve 467

mouse (n = 3) at day -1, 0 and 1, and all mice were then subsequently challenged with 468

P. yoelii 17XL. The parasitemia (B) and survival rate (C) were determined. **p < 469

0.01. 470

471

Figure 4: The frequency and number of GC B cells in the spleens of the 472

ITV-immunized WT and PD-1-/-

mice. Splenocytes were isolated from the immunized 473

WT and PD-1-/-

mice at the indicated time after the final immunization, and both the 474

frequency and number of GC B cells was analyzed by FACS. A, Representative FACS 475

analysis of B220+GL-7

+CD95

+ GC B cells at days 7, 14 and 21. B and C, Statistical 476

analysis of the frequency (B) and number (C) of GC B cells from the ITV-immunized 477

WT and PD-1-/-

mice at days 7, 9, 11, 14 and 21. Three individual experiments were 478

performed. The data are presented as the mean ± SD; ns, no significance; *p < 0.05; 479

**p < 0.01. 480

481

Figure 5: The frequency and number of malaria-specific TFH cells from the 482

ITV-immunized WT and PD-1-/-

mice. Splenocytes were isolated from the immunized 483

WT (n=5) and PD-1-/-

mice (n = 5) at the indicated day after the final immunization, 484

and the TFH cells were analyzed by FACS. A, Representative FACS analysis of 485

CD4+CD11a

+CD49

+CXCR5

+ICOS

+ malaria-specific TFH cells; B and C, Statistical 486

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

+CD49

+CXCR5

+ICOS

+ 487

cells in the immunized WT and PD-1-/-

mice. D, Representative FACS analysis of 488

CD4+CD11a

+CD49

+CXCR5

+Bcl6

+ malaria-specific TFH cells at days 7, 14 and 21. E 489

and F, Statistical analysis of the frequency (E) and number (F) TFH cells 490

(CD4+CD11a

+CD49

+CXCR5

+Bcl6

+) from the immunized WT and PD-1

-/- mice at 491

days 7, 9, 11, 14 and 21. Three experiments were performed. The data are presented 492

as the mean ±SD; *p< 0.05; **p< 0.01. 493

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