PD-1 Deficiency Enhances Humoral Immunity of Malaria InfectionTreatment Vaccine

Taiping Liu,a Xiao Lu,a Chenghao Zhao,a Xiaolan Fu,b Tingting Zhao,b Wenyue Xua

Department of Pathogenic Biology, Third Military Medical University, Chongqing, People’s Republic of Chinaa; Institute of Immunology of PLA, Third Military MedicalUniversity, Chongqing, People’s Republic of Chinab

Malaria infection treatment vaccine (ITV) is a promising strategy to induce homologous and heterologous protective immunityagainst the blood stage of the parasite. However, the underlying mechanism of protection remains largely unknown. Here, wefound that a malaria-specific antibody (Ab) could mediate the protective immunity of ITV-immunized mice. Interestingly, PD-1deficiency greatly elevated the levels of both malaria-specific total IgG and subclass IgG2a and enhanced the protective efficacy ofITV-immunized mice against the blood-stage challenge. A serum adoptive-transfer assay demonstrated that the increased Ablevel contributed to the enhanced protective efficacy of the immunized PD-1-deficient mice. Further study showed that PD-1deficiency could also promote the expansion of germinal center (GC) B cells and malaria parasite-specific TFH cells in the spleensof ITV-immunized mice. These results suggest that PD-1 deficiency improves the protective efficacy of ITV-immunized mice bypromoting the generation of malaria parasite-specific Ab and the expansion of GC B cells. The results of this study provide newevidence to support the negative function of PD-1 on humoral immunity and will guide the design of a more effective malariavaccine.

Although malaria control programs have led to an extensivereduction in malaria incidence and mortality, malaria re-

mains one of the most threatening diseases worldwide. It is esti-mated that 207 million cases and 627,000 malaria deaths occurredin 2012 (1).

A vaccine is regarded as the most cost-effective strategy to pre-vent malaria infection (2). Most malaria subunit blood-stage vac-cines have been designed to induce antibodies (Ab) against a va-riety of surface proteins on the merozoite to block the invasion ofred blood cells (RBCs) (3). However, the invasion of the merozo-ites into red blood cells is controlled by multiple redundant pro-teins (4), and Ab against one or two merozoite surface proteins areunable to effectively prevent the infection of red blood cells withthe malaria parasite (4). Furthermore, most merozoite surfaceproteins exhibit antigenic polymorphism under selective pressure(5). To date, there is no malaria subunit vaccine available world-wide.

In contrast to the subunit malaria vaccine, the malaria infec-tion treatment vaccine (ITV), which involves infection with livemalaria parasites under curative antimalarial drug coverage, hasbeen reported to induce antibodies specific for the merozoite sur-face antigens conserved between heterologous strains but not forthe variant surface antigens (6). ITV induces strong protectiveimmunity against the blood stage of the parasite in animals (7)and humans (8). Interestingly, ITV can also confer cross-protec-tion against the liver stage of malaria by inducing cellular immuneresponses (7). However, the underlying mechanism of protectiveimmunity induced by ITV is still largely unknown.

Follicular helper CD4 T(TFH) cells are characterized by thehigh expression of chemokine receptor CXCR5, programmeddeath 1 (PD-1), lineage-specific transcription regulator Bcl6, SAP(SH2D1A), interleukin-21, and ICOS and are recognized as spe-cialized providers of cognate B cell help (9). Of these characteristicmolecules, PD-1 has been reported to provide modulatory signalsto germinal center (GC) TFH cells, but its function in the modu-lation of humoral immunity remains unresolved. Some evidence

has shown that the blockade of PD-L1 or PD-1 reinforces TFH cellexpansion, increases the number of GC B cells and plasmablasts,and enhances antigen-specific Ab responses (10, 11). However,attenuated humoral immune responses also have been observedafter blockade of PD-1 signaling (12–14). Therefore, the exact roleof PD-1 signaling in the protective immunity of the ITV-immu-nized mice remains unclear.

In this study, we found that PD-1 deficiency greatly improvedthe protective efficacy of ITV-immunized mice against a malariablood-stage challenge. This phenomenon was attributed to theelevated malaria parasite-specific Ab in the immunized PD-1-de-ficient mice. In addition, we also observed increased GC B cellsand the expansion of TFH cells in immunized PD-1-deficient mice.Thus, our data further confirmed the negative effect of PD-1 sig-naling on humoral immunity and shed new light on the design ofeffective malaria vaccine.

MATERIALS AND METHODSMice and Plasmodium parasites. PD-1�/� mice (BALB/c background)were obtained from the Jackson Laboratory (Bar Harbor, ME). Specific-pathogen-free BALB/c mice, at 6 to 8 weeks of age, were purchased fromthe Beijing Animal Institute. All animal protocols were reviewed and ap-proved by the Animal Ethics Committee of the Third Military Medical

Received 12 September 2014 Returned for modification 19 October 2014Accepted 22 February 2015

Accepted manuscript posted online 2 March 2015

Citation Liu T, Lu X, Zhao C, Fu X, Zhao T, Xu W. 2015. PD-1 deficiency enhanceshumoral immunity of malaria infection treatment vaccine. Infect Immun83:2011–2017. doi:10.1128/IAI.02621-14.

Editor: J. H. Adams

Address correspondence to Tingting Zhao, zhaotingting95@163.com, or WenyueXu, xuwenyue@gmail.com.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02621-14

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University Institute of Medical Research. The lethal strain Plasmodiumyoelii 17XL was obtained from MR4 (Malaria Research and ReferenceReagent Resource Center, Manassas, VA) and maintained by intraperito-neal (i.p.) passages in mice.

Immunization and challenge. The immunization schedule was per-formed as previously described (7) with minor modifications. Briefly,mice were intravenously (i.v.) injected with 106 P. yoelii 17XL infectedRBCs (Py-iRBCs) or an equivalent amount of normal RBCs with or with-out 100 �l of 8 mg of chloroquine (CQ; Sigma-Aldrich)/ml diluted insaline daily for 15 days starting from the day of iRBC injection. The micewere maintained for 21 days after the last CQ injection to allow completeelimination of the drug and challenged i.p. with 106 Py-iRBCs.

Adoptive serum transfer assay and CD4� T cell depletion. For serumtransfer, naive BALB/c mice were injected i.v. on days �1, 0, and 1 with 0.2ml of naive mouse serum, ITV-immunized wild-type (WT) serum, orPD-1�/� mouse serum collected 21 days after the last CQ injection, asdescribed previously (15). The mice were challenged with 2.5 � 104 Py-iRBCs on day 0, and parasitemia and the survival rate were determined.For CD4 depletion studies, an anti-CD4-depleting monoclonal antibody(GK1.5 clone, 200 �g per mouse in 200 �l of phosphate-buffered saline[PBS]; BioXcell) or control Ab was injected i.p. on days �1 and 1 (i.e., 20and 22 days after the last CQ injection) according to a previously de-scribed protocol (16). CD4� T cell depletion was verified by stainingblood samples with anti-CD4 (clone RM4-5; eBioscience). The mice werethen challenged with 106 Py-iRBCs on day 0.

Detection of malaria-specific IgG in serum. Sera were collected fromnaive mice, ITV-immunized WT mice, and PD-1�/� mice at 21 days afterthe last CQ injection. Hyperimmune sera were collected from the ITV-immunized WT mice that had recovered from the P. yoelii 17XL infection.The malaria-specific total IgG, IgG1, and IgG2a in the serum were de-tected as previously described (15, 17). Briefly, P. yoelii 17XL-infectedmouse blood was collected, lysed with 0.01% saponin (Sigma-Aldrich) at37°C for 20 min, and sonicated in PBS. Nunc MaxiSorp immunoplates(Nalge Nunc) were coated with parasite antigen at a concentration of 5 to10 �g/ml overnight at 4°C and coincubated with serial dilutions of serafrom the ITV-immunized WT and PD-1�/� mice. Biotin-conjugated an-ti-mouse IgG1 and IgG2a (BioLegend) were added to the plates to detectIgG1 and IgG2a. After a washing step with wash buffer, the plates wereincubated with horseradish peroxidase (HRP)-conjugated anti-mouse

IgG or HRP-conjugated streptavidin (BioLegend), and 3,3=,5,5=-tetram-ethylbenzidine was added (BioLegend). The absorbance at a wavelengthof 450 nm was read using a spectrophotometer.

Flow cytometry analysis of GC B and malaria-specific TFH cells. BothGC B and malaria-specific TFH cells from the naive mice and ITV-immu-nized WT and PD-1�/� mice were analyzed at 7, 9, 11, 14, and 21 days (at21, 23, 25, 28, and 35 days after the start of CQ treatment, respectively)after the last CQ injection. In brief, single-cell suspensions of splenocyteswere prepared and washed in flow cytometry buffer (PBS with 2% fetalcalf serum and 0.05% sodium azide), followed by blocking with anti-mouse CD16/32 (BioLegend). For the GC B cell analysis, 106 cells wereincubated with anti-mouse B220 allophycocyanin (APC; BioLegend),anti-mouse CD95 phycoerythrin (PE; eBioscience), and anti-mouse Tand B cell activation marker (GL-7) fluorescein isothiocyanate (FITC;BioLegend). For the malaria-specific TFH cell analysis, 2 � 106 cellswere incubated with anti-mouse CXCR5 biotin (BioLegend), strepta-vidin-APC (BioLegend), anti-mouse CD4 APC/Cy7 (BioLegend),anti-mouse CD11a percp/Cy5.5 (BioLegend), anti-mouse CD49dFITC (BioLegend), anti-mouse ICOS PE/Cy7 (BioLegend), or anti-mouse Bcl6 PE (eBioscience) after the cells were permeabilized withfixation/permeabilization agent (eBioscience). The cells were then an-alyzed by using flow cytometry.

Statistical analysis. Differences between samples were analyzed usingthe GraphPad Prism version 5.0. Since our data were not confirmed to benormally distributed, nonparametric tests were used to determine thestatistical significance between groups. We use the Mann-Whitney test tocompare two groups and the Kruskal-Wallis test to compare more thantwo groups. P values of �0.05 were considered significant.

RESULTSThe absence of PD-1 greatly enhanced the protective efficacy inthe ITV-immunized mice. To determine whether PD-1-defi-ciency could enhance the protective efficacy in the ITV-immu-nized mice, the parasitemia levels and survival rates of the ITV-immunized WT and PD-1-deficient mice were compared after ablood-stage challenge, as depicted in Fig. 1. The parasitemiacurves of the PD-1-deficient mice were comparable to those ofWT mice, indicating that the PD-1-deficient mice had no intrinsic

FIG 1 The protective efficacy against blood-stage challenge was markedly enhanced in the ITV-immunized PD-1�/� mice. (A) Procedure for the ITVimmunization and challenge. (B and C) Naive or immunized WT (n � 5) and PD-1�/� (n � 5) mice were challenged i.p. with P. yoelii 17XL iRBCs at day 21 afterthe last CQ injection. The parasitemia and survival rate were recorded. The results are representative of three independent experiments. The data are presentedas means � the standard deviations (SD). **, P � 0.01.

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resistance to the malaria parasite. However, compared to the non-immunized mice, the growth of parasite in either ITV-immunizedWT mice or PD-1-deficient mice was greatly suppressed. The peakparasitemia in the immunized WT mice was 10.47% � 0.095%but only 0.017% � 0.029% in immunized PD-1�/� mice at day 4after live P. yoelii 17XL challenge (Fig. 1B) (P � 0.01). Parasiteswere cleared from all immunized mice at day 8 postchallenge, andall of the mice survived (Fig. 1C). These data demonstrate that aPD-1 deficiency could largely augment the protective efficacy ofthe ITV protocol.

Malaria parasite-specific Ab was necessary for the protectiveimmunity of the ITV-immunized mice. To elucidate the mecha-nism of the augmented protective efficacy in the immunized PD-1�/� mice, we first determined the protective immunity of theITV-immunized mice. Both Ab and CD4� T cell responses areessential for controlling the malaria blood-stage development(18). Therefore, sera from either naive mice or ITV-immunizedmice were adoptively transferred to naive mice, and the recipientmice were then challenged with live P. yoelii 17XL. The resultingparasitemia levels of the mice that received naive mouse sera werecomparable to those of naive mice, and all of the mice died. How-ever, the mice that received sera from the ITV-immunized miceserum cleared the parasites at day 18 postchallenge, and all of themice survived (Fig. 2A and B). These data strongly suggest thatantibodies can mediate the protection of ITV-immunized miceagainst malaria parasite challenge.

Next, the CD4� T cells of the immunized WT mice were de-pleted, and the mice were challenged with P. yoelii 17XL. As shownin Fig. 2C and D, no significant difference in the parasitemia orsurvival rate was found between the ITV-immunized mice in-jected with control Ab and those injected with anti-CD4 Ab. Thus,

in contrast to Ab, the data suggest that CD4�T cells are not essen-tial for ITV-immunized mice against the blood-stage challenge.

Elevated malaria-specific Ab greatly contribute to enhancedprotective efficacy in the immunized PD-1�/� mice. Because Abare capable of mediating the protective immune response of theITV-immunized mice, the levels of malaria-specific IgG werecompared between the ITV-immunized WT mice and PD-1�/�

mice. As shown in Fig. 3A, the levels of malaria parasite-specifictotal IgG and isotype IgG2a in immunized WT mice were muchlower than those in the immunized PD-1�/� mice (IgG, P � 0.05;IgG2a, P � 0.05), although no significant difference in the IgG1levels was found between the two types of immunized mice (IgG1,P 0.05). Thus, these data suggest that the augmented protectiveefficacy in immunized PD-1�/� mice was closely associated withthe elevated levels of malaria-specific Ab.

To confirm that the elevated Ab contributed to the improvedprotective immunity of the ITV-immunized PD-1�/� mice, serafrom the ITV-immunized WT mice or PD-1�/� mice were adop-tively transferred to naive mice, and the recipient mice were thenchallenged with P. yoelii 17XL. As a result, the appearance of par-asite in the blood of the mice that received the immunized PD-1-deficient mouse sera was delayed by 2 days compared to that ofmice who received the immunized WT mouse sera (Fig. 3B). Al-though all mice receiving sera from either type of immunizedmouse survived (Fig. 3C), the parasitemia in mice that receivedimmunized PD-1-deficient mouse sera was only 3.38% � 0.69%,i.e., much lower than that of the mice that received sera from theimmunized WT mice (40.86% � 5.22%) at day 8 after live P. yoelii17XL challenge (P � 0.01). Therefore, these data strongly suggestthat the elevated malaria-specific Ab greatly contribute to the en-hanced protective efficacy in the ITV-immunized PD-1�/� mice.

FIG 2 Protective immunity of the ITV-immunized WT mice. (A and B) Sera from the naive mice or ITV-immunized WT mice were adoptively transferred intoeach naive mouse (n � 3) at days �1, 0, and 1. All mice were challenged with P. yoelii 17XL on day 0, and the parasitemia (A) and survival rate (B) weredetermined. (C and D) On days �1 and 1 before the challenge, immunized WT mice (n � 3) were injected with anti-CD4 or control IgG. All of the mice werethen challenged with P. yoelii 17XL on day 0, and the parasitemia and survival rate were monitored. All experiments were performed twice. The data are presentedas means � the SD.

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The frequency and number of GC B cells significantly in-creased in immunized PD-1-deficient mice. To further confirmthe role of PD-1 signaling in the regulation of Ab production, thefrequencies of GC B cells in the spleen were detected at days 7, 9,11, 14, and 21 after the final injection of CQ. As shown in Fig. 4,the frequency and number of GC B cells in both ITV-immunizedWT mice and PD-1�/� mice gradually increased over time. How-ever, the GC B frequency and number of the ITV-immunizedPD-1�/� mice were much higher than those of the ITV-immu-nized mice at days 14 and 21 (P � 0.01), but no significant differ-ence was found either at day 7 or day 9 after the final injection ofCQ. These results suggest that PD-1 deficiency could promote theexpansion of GC B cells in the spleens of immunized WT mice;this conclusion is in agreement with the elevated level of Ab in thesera.

Plasmodium-specific TFH cells expanded in the immunizedPD-1�/� mice. TFH cells can provide help to GC B cells for thegeneration of GCs and long-term protective humoral responses(19, 20). To test whether the increased GC B cells frequency in theITV-immunized PD-1-deficient mice was a result of the expan-sion of TFH cells, the frequency and number of splenic TFH cellswere compared between immunized WT mice and PD-1�/� mice.As described in the previous study (9), we used the activationmarkers CD4, CXCR5, and ICOS and the transcription factor Bcl6to characterize splenic TFH cells. In addition, the coordinated up-regulation of the integrins CD49d and CD11a on antigen-experi-enced CD4� T cells has also been used to identify Plasmodium-specific CD4� T cells (21). Therefore, CD4� CD11a� CD49d�

CXCR5� ICOS� or CD4� CD11a� CD49d� CXCR5� Bcl6� cells

were considered Plasmodium-specific TFH cells in our study (Fig.5A and D).

The frequency and number of TFH cells in both ITV-immu-nized WT mice and PD-1�/� mice gradually decreased over time(Fig. 5B, C, E, and F). The highest TFH cell frequencies and num-bers were observed at day 7 after the final injection of CQ, which isconsistent with previous reports (22, 23), and the frequency andnumber were reduced to the baseline level at day 21. However, thefrequencies and numbers of CD4� CD11a� CD49d� CXCR5�

ICOS� TFH cells or CD4� CD11a� CD49d� CXCR5� Bcl6� TFH

cells from the immunized PD-1�/� mice were 3-fold greaterthan those of immunized WT mice at day 7 after the final injectionof CQ (P � 0.01; Fig. 5). Thus, the increased malaria parasite-specific TFH cells were closely associated with the expansion of GCB cells and elevated Ab in the sera.

DISCUSSION

Due to the failure of malaria blood-stage subunit vaccines, whole-parasite vaccines, such as ITV (7), whole-killed parasites (17), andgenetically attenuated parasites (24, 25), have received more at-tention from researchers in recent years. Understanding the un-derlying mechanism of the whole-parasite vaccine would help inthe design of a more effective malaria vaccine. Here, we foundthat the production of malaria parasite-specific antibodies werecapable of mediating protection of the ITV-immunized mice. In-terestingly, PD-1 deficiency leads to the sterile protection of theITV-immunized mice against the malaria blood stage; this phe-nomenon was correlated to the elevated malaria parasite-specificAb in the serum. In addition, the elevated malaria parasite-specific

FIG 3 Elevated malaria-specific Ab contributed to the enhanced protective efficacy of the ITV-immunized PD-1�/� mice. (A) Three weeks after the finalimmunization, the levels of total IgG, IgG1, and IgG2a in the sera of both immunized WT (n � 5) and PD-1�/� (n � 5) mice were detected by enzyme-linkedimmunosorbent assay. Sera from PBS-immunized mice served as the negative control, and hyperimmune sera served as the positive control. The data arepresented as the log (lg) of the Ab titer. (B and C) Sera from the naive mice, ITV-immunized WT mice, or PD-1�/� mice were adoptively transferred into eachnaive mouse (n � 3) at days �1, 0, and 1, and all mice were then challenged with P. yoelii 17XL. The parasitemia and survival rate were determined. ns, notsignificant; *, P � 0.05; **, P � 0.01.

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Ab was closely associated with the expansion of GC B cells andmalaria parasite-specific TFH cells in immunized PD-1-deficientmice.

We found that the adoptive transfer of ITV-immunized mousesera could delay and reduce the parasitemia after a blood-stagechallenge (Fig. 2), although its effect was short-term likely due tothe clearance of antibodies following binding to the parasites. Ex-cept for the high level of malaria-specific Ab, the possible changedantibody affinity, which was not tested in our study, might alsocontribute to the enhanced protective immunity of ITV-immu-nized mice. However, CD4� T cell depletion prior to the challenge(day 21) did not alter the protection of the ITV-immunized mice,although a great expansion of TFH was observed early (days 7, 9,11, and 14) after the vaccination (Fig. 5). In addition, blockade ofPD-L1 and LAG-3 could promote the differentiation of CD4� TFH

cells and plasmablasts in malaria infection (21). These data showthat TFH cells might provide the specialized help in the generationof GC B cells and Ab at the early postvaccination stage (9) but notat the late stage when both GC B cells and Ab have already formed.Therefore, the protective immunity of the ITV-immunized micewas largely dependent on the malaria parasite-specific Ab, but arole for CD4�T cells to help antibody response during immuni-zation could not be completely excluded.

Although ITV immunization could induce protective immu-nity against the blood stage (6) and the liver stage (7) of the para-site, short-term parasitemia was observed after challenge in bothour study and other studies (6). Thus, vaccinated individuals maystill develop clinical symptoms and may be able to transmit themalaria parasite to mosquitoes after malaria parasite challenge.Interestingly, we found that PD-1 deficiency resulted in the sterileprotection of the ITV-immunized mice (Fig. 1); sterile protectionwould prevent the development of clinical symptom and thetransmission of malaria by vaccinated individuals. This type of

vaccine would greatly contribute to the elimination of malariaworldwide.

Recently, evidence has shown that parasitized erythroblastcould activate CD8� T cells (26). Although CD8� T cells werefound to be dispensable for the protective effect of ITV-immu-nized mice against blood-stage challenge (7), it is protective in theimmunized mice that survived infection with both P. yoelii XNLand, subsequently, P. yoelii 17XL (27). In addition, previous stud-ies showed that PD-1 signaling could induce CD8� T cell anergy,not only in virus infection (28, 29) but also in chronic malariainfection (30). Therefore, the contribution of CD8� T cells to theenhanced protective immunity of the ITV-immunized PD-1-de-ficient mice still could not be completely excluded.

Although recent studies have revealed that PD-1 signaling canalso modulate the T cell-dependent humoral immunity, the re-ports regarding the function of PD-1 signaling in the control ofhumoral immunity remain contradictory (10–14). Here, wefound PD-1 deficiency could significantly elevate the levels of ma-laria parasite-specific total IgG and isotype IgG2a in the serum,and it greatly promoted the expansion of both GC B cells and TFH

cells in the ITV-immunized mice. Serum adoptive-transfer assaysfurther confirmed the negative role of PD-1 signaling in the con-trol of humoral immunity. This is consistent with a negative reg-ulatory role of PD-1 signaling in the regulation of TFH in chronicmalaria infections (21).

PD-1 has two known ligands: PD-L1 and PD-L2. PD-L1 isexpressed on a wider range of cells than PD-L2, but both of themcan be expressed on GC B cells and dendritic cells (31). AlthoughPD-1/PD-L1 or PD-1/PD-L2 signaling has been reported to mod-ulate TFH cells, GC B cells, and Ab (11, 12), the ligand that isinvolved in the humoral immunity of ITV-immunized mice re-mains to be determined. Recently, follicular regulatory T cells(TFR cells) that suppress the GC reaction were identified (32, 33).

FIG 4 Frequency and number of GC B cells in the spleens of the ITV-immunized WT and PD-1�/� mice. Splenocytes were isolated from immunized WT and PD-1�/�

mice at the indicated times after the final immunization, and both the frequency and the number of GC B cells were analyzed by fluorescence-activated cell sorting(FACS). (A) Representative FACS analysis of B220� GL-7� CD95� GC B cells at days 7, 14, and 21. (B and C) Statistical analysis of the frequency and number of GC Bcells from the ITV-immunized WT and PD-1�/� mice at days 7, 9, 11, 14, and 21. Three individual experiments were performed. The data are presented as means � theSD. *, P � 0.05; **, P � 0.01.

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Because FoxP3 is the only marker that distinguishes TFR from TFH,it seems likely that previously identified “TFH cells” with markersof ICOS, CXCR5, and PD-1 could be mixtures of stimulatory TFH

cells and inhibitory TFR cells. Therefore, the effect of PD-1 defi-ciency on TFH and TFR cells in the ITV-immunized mice warrantsfurther investigation.

In conclusion, we demonstrate that malaria parasite-specific Absare capable of mediating the protective immunity of the ITV-immu-

nized mice. Interestingly, PD-1 deficiency could confer sterile protec-tive immunity to the ITV-immunized mice; this is an important stepin the worldwide elimination of malaria. We also provide evidencethat PD-1 signaling could greatly enhance the malaria-specific B cellresponse and the expansion of TFH cells, further supporting the neg-ative role of PD-1 signaling in the modulation of humoral immunity.Thus, our findings not only have implications for the rational designof an effective blood-stage vaccine against malaria parasites through

FIG 5 Frequency and number of malaria-specific TFH cells from ITV-immunized WT and PD-1�/� mice. Splenocytes were isolated from the immunizedWT (n � 5) and PD-1�/� mice (n � 5) at the indicated day after the final immunization, and the TFH cells were analyzed by FACS. (A) RepresentativeFACS analysis of CD4� CD11a� CD49� CXCR5� ICOS� malaria-specific TFH cells. (B and C) Statistical analysis of the frequency and number of CD4�

CD11a� CD49� CXCR5� ICOS� cells in the immunized WT and PD-1�/� mice. (D) Representative FACS analysis of CD4� CD11a� CD49� CXCR5�

Bcl6� malaria-specific TFH cells at days 7, 14, and 21. (E and F) Statistical analysis of the frequency and number of TFH cells (CD4� CD11a� CD49�

CXCR5� Bcl6�) from immunized WT and PD-1�/� mice at days 7, 9, 11, 14, and 21. Three experiments were performed. The data are presented as means� the SD. *, P � 0.05; **, P � 0.01.

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the induction of a robust B cells response but also further our under-standing of the regulatory role of PD-1 signaling in the humoral im-mune response.

ACKNOWLEDGMENTS

This study was supported by the National Science Foundation of China(grant 81271859), the Natural Science Foundation of PLA (CWS12J093),and a major project of PLA (BWS11J041).

We also thank W. Peters and B. L. Robinson from the Malaria Re-search and the Reference Reagent Resource Center for providing P. yoelii17XL.

REFERENCES1. World Health Organization. 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 cen-tury. EMBO Mol Med 6:708 –720. http://dx.doi.org/10.1002/emmm.201403876.

3. Genton B, Reed ZH. 2007. Asexual blood-stage malaria vaccine develop-ment: facing the challenges. Curr Opin Infect Dis 20:467– 475. http://dx.doi.org/10.1097/QCO.0b013e3282dd7a29.

4. Cowman AF, Crabb BS. 2006. Invasion of red blood cells by malariaparasites. Cell 124:755–766. http://dx.doi.org/10.1016/j.cell.2006.02.006.

5. Mu J, Awadalla P, Duan J, McGee KM, Keebler J, Seydel K, McVeanGA, Su XZ. 2007. Genome-wide variation and identification of vaccinetargets in the Plasmodium falciparum genome. Nat Genet 39:126 –130.http://dx.doi.org/10.1038/ng1924.

6. Elliott SR, Kuns RD, Good MF. 2005. Heterologous immunity in theabsence of variant-specific antibodies after exposure to subpatent infec-tion with blood-stage malaria. Infect Immun 73:2478 –2485. http://dx.doi.org/10.1128/IAI.73.4.2478-2485.2005.

7. Belnoue E, Voza T, Costa FT, Gruner AC, Mauduit M, Rosa DS,Depinay N, Kayibanda M, Vigario AM, Mazier D, Snounou G, SinnisP, Renia L. 2008. Vaccination with live Plasmodium yoelii blood-stageparasites under chloroquine cover induces cross-stage immunity againstmalaria liver stage. J Immunol 181:8552– 8558. http://dx.doi.org/10.4049/jimmunol.181.12.8552.

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 tomalaria after administration of ultralow doses of red cells infected withPlasmodium falciparum. Lancet 360:610 – 617. http://dx.doi.org/10.1016/S0140-6736(02)09784-2.

9. Crotty S. 2011. Follicular helper CD4 T cells (TFH). Annu Rev Immunol29:621–663. http://dx.doi.org/10.1146/annurev-immunol-031210-101400.

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. http://dx.doi.org/10.1038/nature07662.

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 humoralimmunity by positively regulating the generation of T follicular helper cells. JImmunol 186:5648–5655. http://dx.doi.org/10.4049/jimmunol.1003161.

12. Good-Jacobson KL, Szumilas CG, Chen L, Sharpe AH, Tomayko MM,Shlomchik MJ. 2010. PD-1 regulates germinal center B cell survival andthe formation and affinity of long-lived plasma cells. Nat Immunol 11:535–542. http://dx.doi.org/10.1038/ni.1877.

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 promotesdistinct effects on T- and B-cell responses in autoimmune arthritis. Eur JImmunol 40:3117–3127. http://dx.doi.org/10.1002/eji.201040690.

14. Kawamoto S, Tran TH, Maruya M, Suzuki K, Doi Y, Tsutsui Y, KatoLM, Fagarasan S. 2012. The inhibitory receptor PD-1 regulates IgA selec-tion and bacterial composition in the gut. Science 336:485– 489. http://dx.doi.org/10.1126/science.1217718.

15. Liu T, Xu G, Guo B, Fu Y, Qiu Y, Ding Y, Zheng H, Fu X, Wu Y, XuW. 2013. An essential role for C5aR signaling in the optimal induction ofa malaria-specific CD4� T cell response by a whole-killed blood-stagevaccine. J Immunol 191:178 –186. http://dx.doi.org/10.4049/jimmunol.1201190.

16. Abes R, Gelize E, Fridman WH, Teillaud JL. 2010. Long-lastingantitumor protection by anti-CD20 antibody through cellular immuneresponse. Blood 116:926 –934. http://dx.doi.org/10.1182/blood-2009-10-248609.

17. Pinzon-Charry A, McPhun V, Kienzle V, Hirunpetcharat C, EngwerdaC, McCarthy J, Good MF. 2010. Low doses of killed parasite in CpG elicitvigorous CD4� T cell responses against blood-stage malaria in mice. J ClinInvest 120:2967–2978. http://dx.doi.org/10.1172/JCI39222.

18. Good MF, Xu H, Wykes M, Engwerda CR. 2005. Development andregulation of cell-mediated immune responses to the blood stages of ma-laria: implications for vaccine research. Annu Rev Immunol 23:69 –99.http://dx.doi.org/10.1146/annurev.immunol.23.021704.115638.

19. Gowthaman U, Chodisetti SB, Agrewala JN. 2010. T cell help to B cells ingerminal centers: putting the jigsaw together. Int Rev Immunol 29:403–420. http://dx.doi.org/10.3109/08830185.2010.496503.

20. Nutt SL, Tarlinton DM. 2011. Germinal center B and follicular helper Tcells: siblings, 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 blockadeof PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodiuminfection. Nat Immunol 13:188 –195. http://dx.doi.org/10.1038/ni.2180.

22. Kang SG, Liu WH, Lu P, Jin HY, Lim HW, Shepherd J, Fremgen D,Verdin E, Oldstone MB, Qi H, Teijaro JR, Xiao C. 2013. MicroRNAs ofthe miR-1792 family are critical regulators of TFH differentiation. NatImmunol 14:849 – 857. http://dx.doi.org/10.1038/ni.2648.

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 directlyon B cells to regulate Bcl-6 expression and germinal center responses. JExp Med 207:353–363. http://dx.doi.org/10.1084/jem.20091738.

24. Ting LM, Gissot M, Coppi A, Sinnis P, Kim K. 2008. AttenuatedPlasmodium yoelii lacking purine nucleoside phosphorylase confer pro-tective immunity. Nat Med 14:954 –958. http://dx.doi.org/10.1038/nm.1867.

25. Aly AS, Downie MJ, Mamoun CB, Kappe SH. 2010. Subpatent infectionwith nucleoside transporter 1-deficient Plasmodium blood-stage parasitesconfers sterile protection against lethal malaria in mice. Cell Microbiol12:930 –938. http://dx.doi.org/10.1111/j.1462-5822.2010.01441.x.

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 eryth-roblasts infected with malaria parasites. Sci Rep 3:1572. http://dx.doi.org/10.1038/srep01572.

27. Imai T, Shen J, Chou B, Duan X, Tu L, Tetsutani K, Moriya C, IshidaH, Hamano S, Shimokawa C, Hisaeda H, Himeno K. 2010. Involvementof CD8� T cells in protective immunity against murine blood-stage infec-tion with Plasmodium yoelii 17XL strain. Eur J Immunol 40:1053–1061.http://dx.doi.org/10.1002/eji.200939525.

28. Maier H, Isogawa M, Freeman GJ, Chisari FV. 2007. PD-1–PD-L1interactions contribute to the functional suppression of virus-specificCD8� T lymphocytes in the liver. J Immunol 178:2714 –2720. http://dx.doi.org/10.4049/jimmunol.178.5.2714.

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 hepatitisC virus core murine model. J Immunol 180:4875– 4884. http://dx.doi.org/10.4049/jimmunol.180.7.4875.

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 ofCD8� T cells drives chronic malaria. Cell Rep 5:1204 –1213. http://dx.doi.org/10.1016/j.celrep.2013.11.002.

31. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. 2008. PD-1 and its ligandsin tolerance and immunity. Annu Rev Immunol 26:677–704. http://dx.doi.org/10.1146/annurev.immunol.26.021607.090331.

32. Chung Y, Tanaka S, Chu F, Nurieva RI, Martinez GJ, Rawal S, WangYH, Lim H, Reynolds JM, Zhou XH, Fan HM, Liu ZM, Neelapu SS,Dong C. 2011. Follicular regulatory T cells expressing Foxp3 and Bcl-6suppress germinal center reactions. Nat Med 17:983–988. http://dx.doi.org/10.1038/nm.2426.

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 controlthe germinal center response. Nat Med 17:975–982. http://dx.doi.org/10.1038/nm.2425.

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