Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to “deactivate” T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4 + T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

The relevance of the interaction between PD-1 and PD-L1 in immunity against cancer and infectious diseases has been extensively studied and therapeutic approaches targeting PD-1 and PD-L1 are in the clinic. In contrast, the role of PD-L2 in modulating immune responses is less clear. The goal of this study was to comprehensively investigate the role for PD-L2 in regulating immunity against malaria. Our studies revealed PD-L2 regulates the PD-1-PD-L1 interaction and has potential to be used as a therapy for malaria.

Field studies in malaria-endemic Mali have shown increased expression of PD-1 on T cells in malaria-infected individuals compared to control subjects, implicating PD-1 in immune evasion (). PD-1-deficient C57BL/6J mice (Pdcd1) rapidly and completely clear non-lethal (P. chabaudi) chronic malaria unlike wild-type (WT) C57BL/6J mice (). Combined blockade of PD-L1 and LAG-3 immuno-inhibitory molecules with antibodies accelerate clearance of acute non-lethal blood-stage malaria (P. yoelii) by improving CD4T cell functions and increasing antibody titers (). Blockade of PD-L1 during lethal malaria (P. berghei) also enhances T cell functions but results in an unfavorable outcome in that model, as T cell hyperactivity promotes cerebral disease (). However, blockade of PD-L2 does not affect immunity against P. berghei malaria (), suggesting that the two PD-1 ligands function differently during malarial infections.

Many studies have shown that pathogens including protozoan parasites such as Plasmodium spp., bacteria, viruses including HIV, and tumor cells exploit the PD-1 pathway to evade the host’s adaptive immunity (). The engagement of PD-1 by its ligands, PD-L1 and PD-L2, normally inhibits T cell functions to induce tolerance and to control the expansion and function of foreign antigen-specific T cells (). PD-L1 and PD-L2 are expressed on a variety of cells, and expression on DCs, as well as other cell types, downregulates T cell immune responses ().

The components of the immune system responsible for eliminating blood stage Plasmodium parasites remain unclear, although antibodies play a key role (). Studies in experimental rodent models of malaria have shown that CD4T helper cell-1 (Th1)-immune responses are critical for protection against acute blood-stage malaria (), while CD8T cells have a role in controlling chronic disease (). Furthermore, several groups have shown that dendritic cell (DC) functions are compromised during malaria (). We have previously shown that naive mice transfused with DCs from mice with non-lethal malaria, but not lethal malaria, survive subsequent infection with lethal malaria (). These studies thus establish that survival from malaria depends on the functional capacity of DCs.

Malaria is a major cause of global morbidity and mortality, caused by parasites of the genus Plasmodium. In 2015 there was an estimated 214 million cases of malaria which led to 438,000 deaths (). Vertebrate hosts become infected by the bite of mosquitoes, which introduce sporozoites that infect hepatocytes, which then release merozoites to infect red blood cells. It is the blood-stage infection that causes the potentially lethal effects of malaria. Much effort has gone into developing vaccines for malaria but success has been limited, highlighting our incomplete understanding of immunity against this disease.

To determine how sPD-L2 exerts its therapeutic effects, we treated P. yoelii YM-infected mice with control Ig or sPD-L2 on days 3 and 5 and collected the spleens at day 7 before the onset of severe clinical symptoms in the control mice. T cells were isolated from spleens and cultured with spleen DCs from naive mice and parasite-specific antigen (MSP1) or peptide (Pb1, EIYIFTNI;) or no additional antigen. Treatment with sPD-L2 increased the number of parasite-specific CD4T cells that could respond to MSP1in culture, with ∼2.7-fold higher numbers of IFN-γ secreting CD4T cells than the mice treated with control Ig, as measured by an ELISPOT assay ( Figure 7 E). Similarly, an in vitro EdU-uptake assay confirmed that sPD-L2-treated mice had higher numbers of parasite-specific T cells which proliferated in response to parasite antigen ( Figure 7 F). However, there was no difference in the number of Tcells between cohorts ( Figure 7 G). Furthermore, the sPD-L2-treated mice also exhibited 6-fold higher numbers of parasite-specific CD8T cells (i.e., that bound MHC tetramer [D] displaying the parasite-specific peptide Pb1;) than the control group ( Figure 7 H). However, there was no increase in IFN-γ secretion ( Figure 7 I) or granzyme B expression ( Figure 7 J) by these cells within 7 days. Similarly, there was no significant increase in serum IFN-γ in response to sPD-L2, ( Figure 7 K). Taken together, these results show that sPD-L2 protects mice from lethal malaria by promoting development of IFN-γ secreting effector CD4T cells, known to be crucial for protection against malaria. Similarly, the increased CD8T cells potentially explained the modest improvement in protection seen in mice treated with sPD-L2 around days 11 to 21 ( Figure 7 D).

To determine the contribution of T cells to sPD-L2-mediated survival from P. yoelii YM malaria, we depleted CD4or CD8T cells in sPD-L2-treated, infected mice. For this experiment, multiple groups of WT mice were infected with P. yoelii YM and treated with sPD-L2 or human IgG (hIg), as in Figure 6 A. These mice were also given CD4or CD8T cell-depleting antibodies or rat Ig on day 1 and every 3–4 days until day 14–18 p.i. Previous studies confirmed that the antibodies used would deplete these cells. All of the infected WT mice that received hIg and rat Ig died or required euthanasia by day 14 ( Figure 7 A and B). In contrast, 75% of the P. yoelii YM-infected mice given sPD-L2 and control rat Ig cleared parasitemia within 30 days and survived > 50 days, when monitoring was stopped ( Figures 7 A and 7B). However, mice were not protected by sPD-L2 if CD4T cells were depleted ( Figures 7 A and 7C) and had to be euthanized due to severity of clinical symptoms. In contrast, depletion of CD8T cells did not significantly affect the protective effect provided by sPD-L2, although these mice had consistently higher parasitemia around days 11–21 than control mice ( Figures 7 A and 7D). Taken together, these findings demonstrate that sPD-L2 can promote survival and parasite control from P. yoelii YM infection through CD4T cells with a possible minor contribution from CD8T cells.

(E–K) T cell responses measured in infected and hIg or sPD-L2-treated mice at day 7 p.i. (E) Numbers of CD4 + T cells that secreted IFN-γ in ELISPOT cultures in response to parasite antigen MSP1 19 in the presence of naive DCs. (F) Numbers of CD4 + T cells that proliferated in cultures in response to parasite antigen MSP1 19 in the presence of naive DCs, measured by incorporation of EdU. (G) Numbers of CD4 + T cells expressing CD25 and FoxP3 per spleen. (H) Numbers of parasite-specific Pb1-tetramer + CD8 + T cells per spleen. (I) Numbers of CD8 + T cells that secreted IFN-γ in cultures in response to parasite peptide Pb1 in the presence of naive DCs (as determined by ELISPOT). (J) Numbers of CD8 + T cells which expressed CD11a a marker of recent activation and intracellular granzyme B. (K) Serum IFN-γ at day 7 p.i. Error bars represent Mean. The data represent two pooled independent experiments. Significance of survival between sPD-L2 + rat IgG treated group with the control group given rat and human Ig or with CD4 + T cell-depleted sPD-L2 treated group was analyzed using Log-rank (Mantel-Cox) test based on data from pooled experiments (N = 8). Significance for assays was analyzed using the non-parametric Mann-Whitney U test based on two-sided tail ( ∗ p < 0.05; ∗∗ p < 0.01). Data represent two independent experiments that obtained similar results.

(A) Survival curves and (B–D) Mean percent parasitemia in WT mice treated with control human IgG (hIg) or sPD-L2 on days 3, 5, and 7 post-infection with P. yoelii YM. Mice were then co-treated with (B) rat Ig, (C) depleting anti-CD4 antibody, or (D) depleting anti-CD8 antibody beginning on day 1 p.i. and every 3–4 days until day 14–18 p.i.

Similarly, 100% of control mice infected with P. berghei developed experimental cerebral malaria symptoms (ECM; Table S2 ) within 8 days ( Figure 6 D) and succumbed to the infection by day 10 ( Figure 6 E). Only 22% of the P. berghei-infected mice treated with sPD-L2 developed cerebral malaria as seen by their ECM scores ( Figure 6 D). Furthermore, the surviving mice controlled the infection for approximately 20 days ( Figures 6 E and 6F), before succumbing 13 days after the last dose of sPD-L2. Additional doses did not improve survival (data not shown). In summary, the administration of multimeric sPD-L2 significantly improved survival from lethal infections and reduced the severity of the clinical symptoms, especially for cerebral malaria.

We hypothesized that multimeric sPD-L2 would outcompete PD-L1 for binding to PD-1 on Th1 cells and thus reduce the suppressive effects of PD-L1 on T cell functions. To test this, we infected WT mice with lethal P. yoelii YM or P. berghei and administered sPD-L2 on day 3, after parasitemia(s) were measurable and then on days 5 and 7 p.i. All WT mice infected with P. yoelii YM and treated with control human IgG (Control Ig) died or had to be euthanized within 10 days ( Figures 6 A and 6B ). Similarly, dimeric PD-L2 did not offer any protection from increasing parasitemia ( Figure S6 A). In contrast, 92% of P. yoelii YM-infected mice (N = 12) treated with sPD-L2 survived and cleared the infection in 25 days with fewer symptoms ( Figures 6 A–6C). All of the surviving mice were rested until day 150 and re-challenged with the same dose of lethal P. yoelii YM malaria (no additional sPD-L2 was administered; Figure 6 A) along with new age-matched, naive control mice (Control Ig-R). All of the mice previously treated with sPD-L2 survived re-infection with no symptoms, and only 4/8 mice showed any parasitemia, as shown by a log scale in the Figure 6 A inset. Within 20 days of re-infection, 80% of these sPD-L2-treated, re-infected mice had completely cleared the infection, as the transfer of 200 μl of blood from these mice to naive mice did not transfer the infection ( Figure S6 B). In comparison, the second set of age-matched control mice succumbed to the infection, confirming the lethality of the parasite used for re-infections ( Figure 6 B). Overall, multimeric PD-L2 could overcome PD-L1 mediated lethality following infection with P. yoelii YM.

(D) Clinical symptom scores, (E) survival, and (F) mean percent parasitemia for a typical course of P. berghei infection in WT mice treated with control (human) IgG or sPD-L2 on days 3, 5, and 7 post-infection (total N = 9 from two independent experiments) are shown. Error bars represent SEM. Significance of survival was analyzed using Log-rank (Mantel-Cox) test. See also Figure S6

(A) Mean percent parasitemia, (B) survival, and (C) clinical symptom scores for a typical course of P. yoelii YM malaria in WT mice treated with Control (human) IgG or sPD-L2 after detectable parasitemia, on day 3 and then days 5 and 7 (total N = 12 from 3 independent experiments) are shown. All surviving mice were rested and after 150 days, re-challenged with the same dose of lethal P. yoelii YM malaria (no additional PD-L2 was administered) along with new age-matched control mice (Control Ig-R). The peak percentage parasitemia during re-infection is highlighted in the inset (N = 8).

We next tested whether multimeric PD-L2 could inhibit PD-L1 binding to PD-1. Using immobilized PD-L1, we compared the binding of PD-1 in buffer alone and in the presence of sPD-L2. PD-1 showed similar binding kinetics to immobilized PD-L1 ( Figure 5 E and Figure S5 B) as in Figure 5 A. However, the interaction between PD-1 and PD-L1 was inhibited by > 80% in the presence of multimeric PD-L2 ( Figure 5 E), confirming that PD-L2 could out-compete PD-L1 binding to PD-1. Taken together, these data provided the proof of concept that sPD-L2 is capable of reducing PD-L1 binding to PD-1 ( Figure 5 ), which could thus in theory prevent PD-L1-mediated lethality in vivo during malaria infection ( Figure 4 A).

We thus hypothesized that large aggregated forms of PD-L2, like those found within a macrocluster on DCs, would compete with PD-L1 for binding to PD-1. To test this hypothesis, we biochemically multimerized this dimeric PD-L2 as described in the supplemental experimental procedures section (hereafter referred to as sPD-L2 as it is a soluble form). Using immobilized PD-1, we found multimeric sPD-L2 had a ∼9-fold higher affinity (16.8 nM) for mouse PD-1 ( Figure 5 D and Figure S5 A) than dimeric PD-L2.

As a previous study reported that PD-L1 and PD-L2 competed for PD-1 binding (), we examined DCs from non-lethal malaria for PD-L1 and PD-L2 co-localization by microscopy. We observed PD-L2 labeling in locally enriched macroclusters, which co-localized with MHC class II in general (arrows in Figure 5 C and Figure S4 A). This explained how PD-L2 was positioned near MHC class II to affect CD3 expression ( Figure 4 ) located within the TCR. Importantly, these macro-clusters of PD-L2 could also co-localize with PD-L1 ( Figure 5 C and Figure S4 B) or were punctate (arrow head in Figure 5 C and Figure S4 C). In contrast, PD-L1 expression was generally punctate, dispersed across the whole cell and co-localized with or adjacent to MHC class II (noted by rust or orange color; Figure 5 C and Figure S4 D). In approximately 30% of DCs, PD-L1 also co-localized with MHC class II into thick ring-like structures ( Figure S4 E). Thus, enriched local concentrations of PD-L2 located within MHC class II synapses, positioned alongside PD-L1, has the potential to compete with PD-L1 for PD-1 binding, to prevent the immunosuppressive functions of PD-L1 on antigen-specific T cells.

To further understand the differential effects of PD-L1 and PD-L2, we undertook biochemical studies using surface plasmon resonance (SPR) technology with dimeric ligands, as a previous study showed PD-L1 has a dimeric structure (). We used recombinant mouse PD-L1 and PD-L2 attached to human immunoglobulin-Fc fusion proteins, which formed dimers due to the intrinsic property of the Fc portion of immunoglobulin, and measured their affinity for PD-1. Using immobilized PD-1, we found that mouse PD-L1 had a Kvalue of 251 nM ( Figure 5 A and Figure S5 A) while PD-L2 ( Figure 5 B and Figure S5 A) had a 1.7-fold higher affinity of 148 nM, in agreement with values reported previously (). PD-L2 had a moderately faster on rate (8.4 × 101/Ms) than PD-L1 (5.6 × 101/Ms) for PD-1 binding but similar off rates (1.1 × 10versus 1.2 × 101/s respectively).

(E) Bar chart comparing binding stability of PD-1 to immobilized PD-L1 and the inhibition of this binding in the presence of 30 μg/ml sPD-L2. A range of concentrations 3.7 μg/mL to 100 μg/mL of PD-1 was passed over immobilized PD-L1 surface. Data represent mean ± SD for three independent experiments. See also Figure S4 for other examples of microscopy and S5 for associated replicate sensograms.

(C) Microscopy of MHC class II, PD-L1 and PD-L2 expression on DCs from spleens of mice infected with a non-lethal parasite with nuclei labeled with DAPI. Arrows indicate co-localizing macroclusters of PD-L2. Arrowheads indicate punctate PD-L2 expression. Open triangles indicate co-localizing microclusters of PD-L1 and MHC class II. Scale bar represents 2 μm.

Binding kinetics of PD-L1-Fc and PD-L2-Fc with immobilized PD-1 receptor. A range of concentrations (A) 3.7 μg/mL to 200 μg/mL of PD-L1-Fc analyte or (B) 3.7 μg/mL to 300 μg/mL PD-L2-Fc analyte was run over a sensor chip surface with immobilized PD-1 receptor. Data represent one of three independent experiments.

Given that PD-L2 expression was associated with survival from malaria, we next examined how PD-L2 co-expression with PD-L1 on DCs could modulate immunity. A previous study has shown that the interaction between PD-L1 on DCs and PD-1 on CD8OTI T cells contribute to ligand-induced T cell receptor (TCR) down-modulation (). We thus investigated whether PD-L2 co-expression with PD-L1 on DCs could inhibit PD-L1-mediated downregulation of TCR and ICOS expression. To do so, we cultured purified DCs and T cells from infected mice (1:5 cells), with antibodies to block PD-1, PD-L1 or PD-L2 functions and examined the T cells after 36 hr for high expression of CD3, a component of the TCR, and high ICOS expression which can indicate T cell activation ( Figures 4 C–4H). Blockade of PD-1-signaling to T cells with anti-PD-1 antibody in DC: T cell cultures significantly increased the expression of CD3 and ICOS, indicating PD-1 signals downregulated expression of these molecules on T cells ( Figures 4 E and 4H). When PD-L1 signals were blocked with antibody, leaving only PD-L2 to function, T cells had significantly increased ICOS and CD3 expression ( Figures 4 F and 4H). In contrast, when PD-L2 was blocked, leaving PD-L1 function intact, there was a significant loss of CD3 and ICOS ( Figures 4 G and 4H). Overall, these findings show that in the context of cells from P. yoelii 17XNL-infected mice, PD-L1 expression on DCs is likely to inhibit T cell activation, whereas PD-L2 appears to promote CD3 and ICOS expression.

We then infected WT and Pdcd1mice (PD-1 deletion on a C57BL/6J background) with lethal P. yoelii YM to confirm that the PD-1 pathway was responsible for the lethality of P. yoelii YM malaria. While 100% of WT mice had to be euthanized by day 10 due to clinical scores ≥ 4, all Pdcd1mice survived ( Figure 4 B) and cleared the infection ( Figure S3 C) confirming that the PD-1 pathway was driving lethality of P. yoelii YM infections. Overall, these studies showed PD-1 and PD-L1 mediate lethality of malaria.

We next undertook DC-transfer studies to establish whether PD-L1 expression on DCs was responsible for lethality of malaria. To do so, WT and Pdcd1lg1mice (PD-L1 deletion on a C57BL/6J background) were infected with lethal P. yoelii YM malaria, DCs isolated at day 7 p.i. ( Figure S3 A) and transferred to naive mice, which were then infected with lethal P. yoelii YM malaria. While 100% of mice given DCs from WT mice had to be euthanized within 10 days due to clinical scores ≥ 4, all mice given DCs from Pdcd1lg1mice survived ( Figure 4 A) and cleared the infection ( Figure S3 B). This transfer study showed that PD-L1 on DCs was mediating lethality as mice given DCs with abundant PD-L1 but little PD-L2 (see Figure 1 F, H and Figure S1 D) did not survive, while all mice given Pdcd1lg1DCs survived.

(H) Scatterplots showing percentages of CD4CD62T cells per well with high CD3 and ICOS expression in replicate wells (N = 3–5) from three independent experiments shown as white, pale blue and darker blue spots. Error bars represent mean. Significance was analyzed using the unpaired t test (one-sided tail) from one of the three experiments (p < 0.05;p < 0.005;p < 0.0005;p < 0.0001). Error bars represent mean. See also Figure S3 for duplicate 4A and 4B experiments.

(C–G) Flow cytometry analysis of CD3 and ICOS expression on CD4 + CD62L lo PD-1 + T cells cultured with DCs (expressing PD-L1 and PD-L2), with and without blockade of PD-1, PD-L1, and PD-L2 compared to control treatment, after 36 hr. Both T cells and DCs were isolated from the spleens of mice infected with P. yoelii 17XNL for 12–14 days. Gates to determine high CD3 or ICOS expression were chosen based on clear double peaks found in anti-PD-L1 cultures.

(B) Survival curves showing Pdcd1 −/− mice are immune to lethal malaria. Groups of 5 WT and Pdcd1 −/− mice were infected with lethal 10 4 P. yoelii YM pRBC and survival monitored every 1–3 days for 50 days in duplicate experiments.

(A) Survival curves showing protection against lethal malaria by DCs without PD-L1 expression. WT and Pdcdlg1 −/− mice were infected with lethal dose of 10 4 P. yoelii YM pRBC, DCs were isolated from infected (drug-cured) mice and ∼1 × 10 7 DCs were transferred to each mouse in groups of four naive mice, in duplicate experiments. After 24 hr, each mouse was infected with 10 4 P. yoelii YM pRBC, and survival monitored every 1–3 days for 50 days.

Overall, our data show that PD-L2 expression is necessary for effective Th1 CD4T cell responses against P. yoelii 17XNL malaria. Given that a higher ratio of PD-L2 to PD-L1 expression on DCs was associated with lower parasitemia and blockade of PD-L2 resulted in reduced CD4Th1 cell responses, we hypothesized that PD-L2 might inhibit PD-L1 functions that were reported to inhibit Th1 cell responses (). Furthermore, PD-L2 blockade caused mortality in mice infected with P. yoelii 17XNL but not P. chabaudi malaria. In alignment, PD-L1 to PD-1-mediated immune suppression was previously shown to be greater during the acute phase of P. yoelii 17XNL () than P. chabaudi malaria (). As such, we conclude that PD-L2-mediated inhibition of PD-L1 to PD-1-mediated immune suppression could explain the different outcomes of PD-L2 blockade between the two infections.

Studies with P. yoelii 17XNL-infected Pdcd1lg2mice also found significantly lower numbers of Tbet-expressing and IFN-γ-secreting, parasite-specific CD4T cells per spleen at day 14 compared to infected WT mice ( Figures S2 G and S2H). Finally, there was no reduction in IFN-γ-secreting, parasite-specific CD8T cells per spleen at day 14 in infected Pdcd1lg2mice or infected mice given anti-PD-L2 blocking antibody compared to infected WT mice ( Figure S2 I).

We next focused on understanding why mice did not survive infection with non-lethal P. yoelii 17XNL when PD-L2 was blocked. We therefore repeated the above blocking experiments and collected spleens at days 7 and 14 for evaluation by multiple immuno-assays. First, CD4T cells were examined for the expression of Tbet, a transcription factor required for effector functions of Th1 CD4T cells, which are known to mediate protection against malaria. T cells were also evaluated for expression of CD62L, a marker found on naive T cells and which also distinguishes central memory (CD62L) from effector memory (CD62L) T cells ( Figure S2 F). Compared to naive mice (day 0; Figure 3 A), there was a significant increase in numbers of Tbet-expressing CD62LCD4T cells per spleen by day 7 ( Figure 3 B; p < 0.0095) in control mice given rat IgG but not mice with PD-L2 blockade ( Figure 3 B; p > 0.05). By day 14, the control mice had 2.2- and 3-fold more Tbet-expressing CD62Land CD62LCD4T cells per spleen, respectively, than the mice given anti-PD-L2 antibody ( Figure 3 C). Similarly, control mice had > 5-fold higher numbers of interferon-γ (IFN-γ)-secreting, parasite-specific CD4T cells at day 14 as measured by responses to parasite antigen MSP1in culture, than mice with PD-L2 blockade ( Figure 3 D). An in vitro EdU-uptake assay confirmed that control mice had higher numbers of parasite-specific CD4T cells which proliferated in response to parasite antigen ( Figure 3 E). However, the concentrations of serum IFN-γ were not affected by PD-L2 blockade ( Figure 3 F). In contrast, mice with PD-L2 blockade had >2-fold more serum interleukin-10 (IL-10) than control mice by day 14 ( Figure 3 G). This result correlated with a significant increase in numbers of regulatory T cells (Treg) per spleen seen with PD-L2 blockade compared to control treated mice ( Figure 3 H).

(H) Mean numbers of CD4T cells expressing CD25 and FoxP3 (regulatory T cells) per spleen. Bar on scatter plots represent median values. The data represent two pooled independent experiments. Significance was analyzed using the non-parametric Mann-Whitney U test based on 2-sided tail (p < 0.05;p < 0.01;p < 0.001). See also Figure S2

(E) Numbers of CD4 + T cells that proliferated in cultures in response to parasite antigen MSP1 19 in the presence of naive DCs, measured by incorporation of EdU.

(D) Numbers of CD4 + T cells that secreted IFN-γ in an ELISPOT culture in response to parasite antigen (MSP1 19 ) in the presence of naive DCs.

To further explore the role of PD-L2 in protection against another non-lethal infection, we infected WT mice with non-lethal P. chabaudi malaria and treated with either anti-PD-L2 or rat IgG ( Figure 2 C and Figure S2 E) as for P. yoelii 17XNL experiments. Mice from both groups survived but blockade of PD-L2 significantly increased parasitemia during the acute infection (day 8; note log scale), led to generally higher parasitemia during the chronic phase of infection (> day 21) and delayed parasite clearance by 4 days (arrow indicates parasite clearance in rat IgG-treated mice; Figure 2 C). Overall, these protection and survival studies showed that PD-L2 expression was required for better control of non-lethal malarias and survival from P. yoelii 17XNL malaria.

To confirm this observation, we next blocked PD-L2 with a monoclonal antibody when parasites became detectable in the blood. For this experiment, WT mice were infected with P. yoelii 17XNL and given either anti-PD-L2 or control rat immunoglobulin G (IgG), 4 days p.i. and every 3–4 days until day 14–18 p.i. All WT mice that received rat IgG survived and cleared the infection within 32 days ( Figure 2 B and Figure S2 C). In contrast, 100% of infected mice that were given the PD-L2 blocking antibody died or were euthanized by day 19, due to severe symptoms ( Figure S2 D), although the degree of parasite control was similar between groups ( Figure 2 B and Figure S2 C). This was in contrast to Pdcd1lg2mice, which had significantly higher parasitemia after day 13 ( Figure 2 A) suggesting either that the antibody did not completely inhibit function, or that 4 days of PD-L2 function, before blockade, partially improved immunity.

To determine the contribution of PD-L2 to the control of malarial parasites, we next examined the outcome of P. yoelii 17XNL infection in Pdcd1lg2mice (PD-L2 deletion on a C57BL/6J background) () compared with WT mice. All WT mice cleared the infection within 27 days ( Figure 2 A). However, the Pdcd1lg2mice had higher parasitemias than WT mice after day 13, and all of these mice died or had to be euthanized by day 19 ( Figure 2 A and Figure S2 A) due to clinical scores ≥ 4 ( Figure S2 B). Thus, PD-L2 expression is required for parasite control and survival from infection with P. yoelii 17XNL.

(A–C) Mean percent parasitemia for a typical course of P. yoelii 17XNL malaria in (A) Pdcd1lg2and WT mice (N = 4) or (B) WT mice treated with rat IgG or anti-PD-L2 blocking antibody (N = 5), and (C) mean percent parasitemia (on a log scale) for a typical course of P. chabaudi malaria in WT mice treated with rat IgG or anti-PD-L2 blocking antibody (N = 5) are shown. Arrow indicates parasites were cleared 4 days earlier in rat IgG than anti-PD-L2-treated mice. Data represent one of two independent experiments that obtained similar results. Significance at certain time points were analyzed using the non-parametric Mann-Whitney U test based on two-sided tail. Error bars represent SEM (p < 0.05;p < 0.005). See also Figure S2

We examined surface expression of PD-L1 and PD-L2 on DCs from the spleen, which has been shown to be a major site of parasite killing and regulation of parasite-specific immune responses in mice (). Approximately 70% of CD11cDCs in the spleens of naive mice expressed PD-L1 and this percentage increased in P. berghei and P. chabaudi-infected mice but not during lethal or non-lethal P. yoelii infections ( Figure 1 F). PD-L1-expressing DCs did show increases in surface expression (mean fluorescence intensity; MFI) of PD-L1 following all four malarial infections compared to DCs from naive mice, with non-lethal P. chabaudi-infected mice showing the greatest increase ( Figure 1 G and Figures S1 A–S1F). In contrast, <5% of splenic DCs from naive mice expressed PD-L2. This differed from human blood DCs that predominantly expressed PD-L2, which most likely reflects their different origins from blood and spleen. Furthermore, the percentages of PD-L2DCs increased during all malarial infections with greater percentages found in mice with non-lethal than lethal malaria ( Figure 1 H and Figures S1 A–S1E). The MFI of PD-L2 expression on PD-L2DCs also increased in mice infected with all but P. berghei parasites, compared to DCs from naive mice ( Figure 1 I and Figures S1 A–S1F). Finally, CD11cDCs from lethal and non-lethal P. yoelii malaria showed similar increases in the amounts of PD-L1 and PD-L2 mRNA ( Figure S1 G) suggesting that the difference in PD-L2 between these parasites ( Figure 1 H) is dependent on post-transcriptional regulation or protein localization. Of note, DCs from lethal and non-lethal P. yoelii infections had the same surface expression of PD-L1 and PD-L2 and mRNA but differed in percentages of PD-L2and not PD-L1DCs. Overall, the results from all infections are consistent with a hypothesis that a higher percentage of PD-L2DCs correlates with a favorable disease outcome.

To understand the biological relevance of these data, we next investigated four mouse models of malaria. We chose four different species and strains of Plasmodium that infect mice, with each showing distinct biology and pathogenicity. When WT mice were infected with non-lethal P. yoelii 17XNL or P. chabaudi, and the blood was examined every 1–3 days for parasites, the infection progressed at different rates, but both groups cleared the infection within ∼30 days ( Figure 1 D). In contrast, WT mice infected with P. yoelii YM or P. berghei ANKA showed severe but distinct disease courses ( Figure 1 E; monitored as per Tables S1 and S2 ). P. berghei parasitemia is low compared to P. yoelii YM infections because P. berghei-infected RBC sequester from the blood into deep tissues including the brain, leading to lethal cerebral disease. However, all P. yoelii YM and P. berghei-infected mice had to be euthanized within 10 days when the clinical score was ≥4 ( Tables S1 and S2 ).

To determine whether PD-L1 and PD-L2 influenced malarial immunity, we infected seven malaria-naive, healthy human volunteers with 1,800 P. falciparum infected red cells (pRBC) and their blood was examined before and 7 days after challenge. We examined DCs, defined by CD11c expression, in view of their important role in malaria pathogenesis () and because PD-L1 and PD-L2 on DCs can downregulate immune responses by T cells (). In all seven volunteers, ∼90% of DCs expressed PD-L1 before infection, and there was no significant change to the percentage of DCs expressing this ligand by day 7 of infection ( Figure 1 A). In contrast, while ∼80% of DCs also expressed PD-L2 before infection, 5 of 7 individuals showed a significantly reduced (17%–57%) percentage of PD-L2DCs at day 7 of infection ( Figure 1 B). Notably, we observed a significant inverse correlation between parasitemia and the ratio of percentage PD-L2 to PD-L1 expression on DCs at day 7 post-infection (p.i.) ( Figure 1 C). Overall, contrary to the generally perceived role of PD-L2 as an immune inhibitor, we observed that higher frequencies of PD-L2-expressing DCs were associated with lower parasitemia in volunteers after infection with P. falciparum.

(H) Percentage of total CD11cDC expressing PD-L2 and (I) MFI of surface PD-L2-expression on PD-L2CD11cspleen DCs from naive and infected mice (D7 p.i.). Bars on scatter plots represent mean value. Significance between matched D0 and D7 human samples was analyzed by Wilcoxon matched-pairs signed rank test. Significance between multiple groups was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. (p < 0.05;p < 0.01;p < 0.001;p < 0.0001 are for comparisons between groups). Data for (F) and (H) represent pooled independent experiments in which similar results were obtained. See also Figure S1 for flow cytometry profiles and MFI in duplicate experiment. Data represent two independent experiments that obtained similar results.

(E) Mean percent parasitemia for typical courses of infection in mice infected with lethal P. yoelii YM or P. berghei and monitored for 10 days. Error bars represent SEM (N = 4–8).

(D) Mean percent parasitemia for typical courses of infection in mice infected with non-lethal P. chabaudi or P. yoelii 17XNL malaria and monitored for up to 40 days.

(C) Plot showing number of parasites/ml blood versus ratio of %PD-L2: %PD-L1 DC. R101 to R108 represents each volunteer. The p value is testing the null hypothesis that the overall slope is zero.

(A–C) Seven healthy human volunteers were inoculated with P. falciparum and blood examined for percentage of CD11c + DC expressing (A) PD-L1 and (B) PD-L2, before (D0) and 7 days (D7) after infection.

Discussion

This study has shown that PD-L2 is essential for establishing effective Th1 CD4+ T cell immunity, which protects against lethal malaria. First, we found that PD-L2 but not PD-L1 expression on human blood DCs changed significantly in response to P. falciparum infections. Unexpectedly, a higher ratio of PD-L2 to PD-L1 expressing-DCs correlated with reduced parasitemia. Mechanistic studies utilized four mouse models of malaria to understand the biological significance of changes in PD-L2 expression. These studies found that while PD-L1 expressed by DCs attenuated immune responses, PD-L2 co-expression regulated immune responses by (1) inhibiting the binding of PD-L1 with PD-1 and (2) increasing CD3 and ICOS expression on T cells. These two functions were so significant, that administration of soluble multimeric PD-L2-Fc fusion protein alone was sufficient to mediate survival of mice from patent lethal malaria and mice then survived re-infections without additional sPD-L2. Thus, we have identified a layer of immune regulation of the PD-L1 with PD-1 interaction and shown a therapeutic strategy for malaria and other diseases susceptible to immune checkpoint modulation.

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Housseau F. Cooperative B7-1/2 (CD80/CD86) and B7-DC costimulation of CD4+ T cells independent of the PD-1 receptor. It is generally believed that PD-L1 and PD-L2, which cannot bind PD-1 simultaneously (), will have the same immuno-inhibitory outcome. However, we have demonstrated that multimeric PD-L2 can out-compete PD-L1 for PD-1-binding. To understand how PD-L2-PD-1 binding might influence immune responses, we compared the effects of selectively blocking PD-L1 and PD-L2 expressed by DC on previously activated T cells in culture by examining CD3 and ICOS expression. We found that blocking PD-L2 signals (PD-L1 function remains) reduced CD3 and ICOS expression while blocking PD-L1 signals (PD-L2 function remains) improved ICOS and CD3 expression. This is consistent with a higher ratio of PD-L2: PD-L1 expression on DCs initiating protective immunity. One crucial point of difference between PD-L1 and PD-L2 is that PD-L1 not only inhibits T cell immunity by binding to PD-1, but can also bind to CD80 (), which can also preferentially recruit CTLA-4 into the immunological synapse (IS) (). Thus, PD-1-PD-L1 binding might also co-recruit CD80 (and CTLA-4) into the IS. In contrast, preferential recruitment of PD-1 by PD-L2 might exclude CD80 (and CTLA-4) recruitment into TCR clusters, to reduce the inhibitory microenvironment surrounding the TCR while recruiting CD3 and ICOS. Therefore the effects of PD-L2 binding PD-1 can be vastly different to the PD-L1 with PD-1 interaction as shown by our study. Furthermore, blocking PD-L1 and leaving PD-L2 free to bind PD-1 increased ICOS and CD3 expression compared to PD-1 blockade. This suggests that PD-L2 does not act merely to compete with PD-L1, but could have an additional stimulatory action via a separate receptor, as previously suggested ().

Remarkably, we showed that three doses of multimeric PD-L2 given to mice with lethal malaria supported the development of robust CD4+ T cell responses that mediated survival from P. yoelii YM infection. These mice then survive re-infections after 150 days with no requirement for additional PD-L2, showing that sPD-L2 administration allowed the development robust long-term protective immunity. In fact, 80% of re-infected mice developed immunity effective enough to completely clear the lethal parasites without patent parasitemia, highlighting that sPD-L2 supports the development of complete immunity. Consequently, sPD-L2 has potential to treat acute infections such as malaria or other diseases where PD-L2 has been downregulated during the immune response, and promote the development of an effective memory response, which is protective against later exposure.