PD -1 deficiency Enhances Humoral Immunity of Malaria ......2015/02/24 · 34 ITV -immunized mice...
Transcript of PD -1 deficiency Enhances Humoral Immunity of Malaria ......2015/02/24 · 34 ITV -immunized mice...
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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
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Running title: PD-1 deficiency and humoral immunity 10
These authors declare that there is no conflict of interest. 11
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# 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:
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
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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
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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
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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
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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
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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
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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
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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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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
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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-/-
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mice. 219
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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
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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
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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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