Control of Toxoplasma reactivation by rescue of dysfunctional CD8+ T-cell response via PD-1–PDL-1 blockade

Rajarshi Bhadraa, Jason P. Gigleya, Louis M. Weissb, and Imtiaz A. Khana,1

aDepartment of Microbiology, Immunology, and Tropical , George Washington University, Washington, DC 20037; and bDepartment of Medicine and Pathology, The Albert Einstein College of Medicine, Bronx, NY 10461

Edited by Jitender P. Dubey, US Department of Agriculture, Beltsville, MD, and approved April 26, 2011 (received for review October 13, 2010)

In this study, we document that differentiation interaction with its receptor PDL-1 not only augmented poly- + and reactivation are mediated by systemic CD8 T-cell dysfunction functional CD8 T-cell response, but it also inhibited parasite during chronic infection. We demonstrate that CD8+ T-cell exhaus- recrudescence and prevented the mortality of chronically infec- + tion occurs despite control of parasitemia during early-chronic ted mice in a CD8 T-cell–dependent manner. To the best of . During later phases, these cells become exhausted, our knowledge, this report of parasite reactivation caused by + leading to parasite reactivation and mortality. Concomitant with PD-1 mediated CD8 T-cell dysfunction is unique. increased CD8+ T-cell apoptosis and decreased effector response, this dysfunction is characterized by a graded elevation in expres- Results sion of inhibitory receptor PD-1 on these cells in both lymphoid Toxoplasma-Infected Mice Show Increased Tachyzoite-Specific Gene + and nonlymphoid tissue. Blockade of the PD-1–PDL-1 pathway Expression with Concomitant Decrease in CD8 T-Cell Response During reinvigorates this suboptimal CD8+ T-cell response, resulting in Chronic Toxoplasmosis. Humans are most commonly infected control of parasite reactivation and prevention of mortality in orally with Type II strains of Toxoplasma (11). To establish the fi chronically infected animals. To the best of our knowledge, this loss of immune control during chronic infection, we rst wanted report is unique in showing that exposure to a persistent patho- to indentify the kinetics of long-term survival in B6 mice infected IMMUNOLOGY gen despite initial control of parasitemia can lead to CD8+ T-cell orally with type II strain (ME49) cysts. Most of these animals dysfunction and parasite reactivation. succumb to infection after 7 wk of challenge (Fig. 1A). Host innate and adaptive immunity are thought to cause differentia- tion of tachyzoites into chronic slow growing bradyzoites (12). recrudescence | Toxoplasma gondii | adaptive immunity Because encysted parasites can persist for life and are not be- lieved to be associated with disease pathology, several studies oxoplasmic encephalitis (TE) from reactivation of chronic have postulated that rather than an incidence of parasites, which Toxoplasma infection affects 24% to 47% of HIV-infected remain primarily encysted in chronically infected brains, the T – Toxoplasma-seropositive patients in the United States (1 3). Al- degree of bradyzoite-tachyzoite interconversion is a better hall- though the incidence of TE among AIDS patients declined mark of disease reactivation (6, 13, 14). Therefore, we assayed threefold in the United States because of prophylactic treatment fi fi parasite reactivation by changes in parasite stage-speci c gene and highly active antiretroviral therapy, it still remains a signi - expression. We measured the transcription of the tachyzoite cant problem in developing countries (4, 5). Hence, understanding stage-specific Enolase-II (ENO-2) and the bradyzoite stage-spe- the mechanisms responsible for parasite reactivation in chroni- cific Enolase-I (ENO-1), two genes that have been previously cally infected hosts is critical for evolving strategies for the de- shown to change in very high levels (15–17). Concomitant with velopment of immunotherapeutic agents against the parasite. poor survival of Toxoplasma gondii-infected mice during the late- In the intermediate host, Toxoplasma undergoes stage conver- chronic phase, increased ENO-2 expression and decreased sion between the rapidly replicating tachyzoite that is thought ENO-1 expression was noted at week 7 postinfection, suggesting to be responsible for acute toxoplasmosis and the slowly repli- parasite reactivation (Fig. 1B). Interestingly, we observed that cating, relatively quiescent, primarily encysted bradyzoite stage expression of both ENO-2 and ENO-1 occurs at week 10, in- that can persist possibly for life (6). Effective CD8+ T-cell re- dicating that despite limited tachyzoite to bradyzoite differenti- sponse, critical for control of both acute and chronic Toxoplas- ation, the parasites are still undergoing reactivation (i.e., a mosis in susceptible mice strains paradoxically do not ensure their bradyzoite to tachyzoite conversion) (Fig. 1B). To verify that long-term survival (7). Poor long-term survival of Toxoplasma- ENO-2 and ENO-1 corresponded to the tachyzoite and brady- infected B10 mice is associated with reduced number of in- T. gondii tracerebral T cells and TE development (8). Additionally, de- zoite stage of , the gene expression of the parasite were pletion of CD8+ T cells rather than CD4+ T cells had a greater examined again in vivo using established models of acute and effect on mortality of chronically infected animals (9). Taken to- chronic infection. Mice were infected with Type I RH strain gether, these studies suggest that although critical for preventing (lethal acute infection) or Type II ME49 strain (chronic) (18, parasite reactivation, CD8+ T-cell responses during late-chronic 19). Some of the ME49-infected animals were treated with drugs known to control reactivation of chronic infection (18, 19). As infection are incapable of preventing mortality in infected mice. fi However, the mechanism underlying this apparently ineffective expected, RH-infected mice exhibited high tachyzoite-speci c CD8+ T-cell response during late-chronic infection has not been gene expression (SAG-1, ENO-2) and minimal bradyzoite-spe- studied and it remains to be determined if this defect is responsible for loss of parasite control. We therefore tested whether CD8+ T cells during late-chronic toxoplasmosis become dysfunctional, as Author contributions: R.B., J.P.G., and I.A.K. designed research; R.B., J.P.G., and L.M.W. reported in some viral infections (10). performed research; L.M.W. contributed new reagents/analytic tools; R.B., J.P.G., L.M.W., This article is unique in demonstrating that CD8+ T cells and I.A.K. analyzed data; and R.B., J.P.G., L.M.W., and I.A.K. wrote the paper. during later phases of chronic toxoplasmosis are unable to mount The authors declare no conflict of interest. a potent anamnestic response, a hallmark of robust adaptive This article is a PNAS Direct Submission. + immunity. Moreover, CD8 T cells from these mice exhibit a 1To whom correspondence should be addressed. E-mail: [email protected]. progressive increase in expression of inhibitory molecule PD-1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with concomitant parasite reactivation. In vivo blockade of PD-1 1073/pnas.1015298108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1015298108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 fi A 100 stage-speci city of the above markers using Type II parasites 80 maintained in vitro under tachyzoite- or bradyzoite-inducing 60 conditions (20). Immunofluorescent microscopic analysis showed 40 fi 20 that stage-speci c protein expression corresponded with our

Percent Survival Percent 0 stage-specific transcript profile (Fig. S1). 0 50 100 150 Days post-infection Tissue cysts in brain occur almost exclusively in neurons and not

ENO-2 ENO-1 in leukocytes during the chronic phase (21). Because a recent B 1.5 p=0.0006 2.5 study has shown that leukocytes can be used for parasite dissem- p=0.025 0.8 2.0 p=0.0135 ination during the acute phase (22), we examined these cells for 0.6 1.5 parasitization as a further readout of reactivation. To character- 0.4 1.0 ize, whether parasite reactivation increased the frequency of

Relative Expression 0.2 0.5 Toxoplasma-infected leukocytes, mononuclear leukocyte-gated 3 5 7 10 3 5 7 10 Weeks post-infection blood and brain cells were examined for parasites by flow cytom- etry. Although minimal infection of blood and brain leukocytes Early Non-Moribund Moribund was noted during the early-chronic phase, high infection rates C Chronic Late chronic Late chronic Week 4-5 Week 7-9 Week 7-13 were observed during the late-chronic period, particularly in 39.1 0.0 32.8 0.1 17.7 0.2 moribund mice (Fig. 1C). Similar to observations noted by Courret et al. (22), the level of parasite infection was much higher in cir- 0.4 7.3 25.8 27.3 0.0 12.0 0.0 9.9 0.1 culating leukocytes vis-à-vis brain leukocytes assayed at the same CD8 time. It has been speculated that this may be because of multiple

Blood Brain Blood populations of nonleukocytic brain lineages that are differentially 6.4 29.1 42.0 T. gondii permissive to T. gondii in the brain (22). Next, we assessed if Spleen Brain reactivated parasites preferentially infected specific leukocyte D 12.0 5.0 subsets in a site-dependent manner. Irrespective of tissue, the majority of parasitized leukocytes corresponded to a myeloid

4 − 6 hi + + 9.0 2.5 phenotype (CD3CD19NK1.1 CD11b F4/80 GR1 )(Fig. S2).

+ x 10 x

x 10 x Taken together, our data suggest that during the late-chronic of CD8 T cells T CD8 of Absolute Number Number Absolute 6.0 0.0 phase, parasite-stage conversion results in reactivation of disease. 0 3 5 7 10 0 3 5 7 10 + Weeks post-infection As Toxoplasma infection induces a robust CD8 T-cell im- munity that is known to be critical for long-term protection, it is E Gating Strategy enigmatic that this potent immune response failed to prevent recrudescence of infection (9). By measuring the kinetics of CD8+ T-cell immunity we demonstrate that CD8+ T-cell re-

CD90.1

CD8 sponse peaked at weeks 3 to 5 postinfection followed by gradual - Gated on CD8 + CD90.1 T cells contraction (Fig. 1D). Concomitant with this process, reduced Naive Week 5 Week 7 + + 0.7 0.0 4.8 11.4 4.1 6.2 frequency of splenic and brain IFN-γ Granzyme B and IFN- − γ Granzyme B+ CD8+ T cells was noted (Fig. 1E). + 0.5 43.8 31.5 One of the quintessential features of robust long-term CD8 0.9 4.2 3.5 1.7 T-cell immunity is the generation of a rapid response upon IFNγ secondary challenge (23). To verify the potency of secondary response, Toxoplasma-infected animals were rechallenged during 36.6 17.0 Granzyme B the early-chronic and late-chronic phases and assessed for cy- Brain Spleen Brain cling CD8+ T cells (Fig. 1F). Mice rechallenged during the early- chronic phase demonstrated a significant increase in absolute F 2.5 p=0.0031 number of proliferating splenic CD8+ T cells over unchallenged +

6 2.0 mice (Fig. 1F). In contrast, no significant change in cycling CD8

+ 10 x 1.5 T cells was noted in rechallenged animals during the late-chronic phase. These data clearly demonstrate that a potent recall re- + 1.0 +

Week 5 Week 7 sponse, a hallmark of robust CD8 T-cell immunity, is com- CD8 Ki-67 Splenic T cells T Splenic Ki-67 CD8 Absolute Number of of Number Absolute No Rechallenge Rechallenge promised during later stages of chronic toxoplasmosis.

Fig. 1. Parasite reactivation during late-chronic T. gondii infection corre- + lates with poor CD8+ T-cell response. (A) C57BL/6 mice were infected with 10 CD8 T Cells Up-Regulate PD-1 During Chronic Toxoplasmosis. The ME49 cysts orally and survival was monitored on a daily basis. (B) Relative role of PD-1 in mediating dysfunction in chronic parasitic models expression of ENO-1 and ENO-2 at weeks 3, 5, 7, and 10 postinfection in the has not been extensively addressed (24, 25). Although PD-1 ex- brain was computed for each sample. Transcript levels at day 10 post- pression during toxoplasmosis has been recently reported (26), + inifection was taken as 1. (C) T. gondii-infected cells in the brain and blood its role in mediating CD8 T-cell dysfunction or pathogenesis as leukocyte-gated samples were assayed by flow cytometry. (D) The absolute shown in chronic viral models like lymphocytic choriomeningitis number of CD8+ T cells at weeks 3, 5, 7, and 10 postinfection. (E) IFN-γ and virus (LCMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Granzyme B production by CD8+ T cells was evaluated in splenocytes or brain and HIV (27), is currently unknown. Analysis of CD8+ T cells cells from infected mice in presence Toxoplasma lysate antigen. (F) At weeks revealed a progressive increase of PD-1 expression and fre- 5 and 7 after primary infection, mice were rechallenged orally with 10 cysts quency during the chronic stage of parasite infection (Fig. 2). Of + and animals were killed 4 d later. The absolute number of cycling CD8 the two PD-1 ligands, PDL-1 and PDL-2, the mean fluorescence T cells was evaluated in the spleen during early-chronic and late-chronic intensity (MFI) of the former but not the latter increased during infection with or without rechallenge. The data represent two experiments the course of chronic infection (Fig. S3). with at least four mice per group. Error bars represent SD throughout. Because increased PD-1 MFI correlated with decreased fre- quency of splenic IFN-γ+CD8+ T cells, we hypothesized that hi + fi PD-1 CD8 T cells would have reduced capacity to release ci c gene expression (BAG-1, ENO-1)(Fig. S1). In contrast, IFN-γ. To address this theory, we measured the proportion of drug-treated mice showed reduced SAG-1 and ENO-2 transcript IFN-γ–producing cells within PD-1hiCD8+ and PD-1intCD8+ T- levels and increased BAG-1 and ENO-1 expression vis-à-vis un- cell population. No significant difference in cytokine-positive treated ME49 infected mice. Next, we further confirmed the cells between the two populations was noted (Fig. S4). Although

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1015298108 Bhadra et al. Downloaded by guest on September 27, 2021 Spleen Liver Brain be attributed to attenuated inflammation or antigen load in drug- 1.3 0.7 treated mice, which can result in suboptimal development or maintenance of Granzyme B-expressing short-lived effector sub- 10.5 2.2 14.2 set (31, 32). Combined, these data suggest that control of infection during the early phases of infection may be critical for minimizing 10.2 2.7 11.4 induction of CD8 dysfunction during the chronic phase.

Blockade of PD-1–PDL-1 Pathway Reinvigorates CD8+ T-Cell Response. 22.4 4.3 23.5 + % of maximum of % Next, we addressed if a dysfunctional CD8 T-cell response could be revived by blocking the PD-1–PDL-1 pathway. To ad- 20.4 4.3 29.5 dress this, animals were administered a nondepleting anti–PDL- 1 antibody starting at week 5 postinfection for a period of 2 wk

Week 10 Week 7 Week 5 Week 3 Naive Week 5 Week 7 Week 10 Week + PD-1 and, thereafter, the CD8 T-cell response in the spleen, liver, and brain were quantified (33). Although significant increase in Fig. 2. PD-1 is expressed at high levels on CD8+ T cells in both lymphoid and absolute number of CD8+ T cells was noted in all of the tissues nonlymphoid tissue during late-chronic Toxoplasmosis. PD-1 expression on + examined, the most dramatic increase occurred in brain (Fig. CD8 T cells from tissues (spleen, brain, and liver) was measured at weeks 3, 3A). To determine if this increase was because of enhanced 5, 7, and 10 postinfection. Filled histogram represents staining with isotype- proliferation, we measured Ki-67 expression in the CD8+ T cells. control antibody. The data represent three experiments with at least four Unlike the spleen and liver, we were unable to detect increased mice per group. frequency of cycling CD8+ T cells in anti–PDL-1 treated groups in the brain (Fig. 3B). Taken together, these data suggest that + – this finding suggests that PD-1 expression does not directly one of the mechanisms for increased brain CD8 T cells in anti regulate IFN-γ production in CD8+ T cells, the role of other PDL-1 treated mice is not in situ proliferation but possibly in- creased recruitment of these cells to the tissue. factors, such as the extent and duration of PD-1 cross-linking and γ frequency of PD-1–PDL-1 engagement in regulating CD8+ T- As IFN- production (34) and cytotoxicity (35) are known to – γ be critical for control of toxoplasmosis, we examined if blockade cell mediated IFN- production, cannot be ruled out. Irre- – + spective of the time point, very few PD-1lo cells produced IFN-γ, with anti PDL-1 augmented these functions within the CD8 T- + cell population. Anti–PDL-1 treatment increased the frequency indicating that an overwhelming majority of CD8 T cells in this + + − + + of IFN-γ Granzyme B and IFN-γ Granzyme B CD8 T cells IMMUNOLOGY subset are naive. Although we did not observe a significant dif- + + hi in the spleen and brain, as well as ex vivo cytotoxicity of splenic ference in frequency of IFN-γ CD8 T cells between the PD-1 CD8+ T cells (Fig. 3 C and D). We then measured, if anti–PDL-1 and PD-1int CD8+ T-cell subsets, we hypothesized that the re- treatment decreased the frequency of PD-1hi cells. Surprisingly, duced frequency of splenic IFN-γ+CD8+ T cells during late- hi PD-1–PDL-1 blockade caused a further increase in the fre- chronic infection could be because of apoptosis of PD-1 cells. As quency of PD-1–expressing CD8+ T cells (Fig. 3E). Because we recent studies have suggested that apoptosis can be coupled to demonstrated that high PD-1 expression was associated with cell-cycle progression (28, 29), we incubated splenocytes from apoptosis, we determined if rescue of CD8+ T-cell response by week 7-infected mice for 5 h at 37 °C and stained for CD8, PD-1, PD-1–PDL-1 blockade was because of reduced apoptosis. To Ki-67, and active caspase-3, the active form of the apoptosis-re- hi + address this under steady-state conditions without confounding lated cysteine peptidase. Although cycling PD-1 CD8 T cells effects of proliferation and graded PD-1 increase, we evaluated exhibited a higher expression of active caspase-3 than noncycling + hi + int/lo apoptotic CD8 T cells in splenocytes from chronically infected PD-1 CD8 T cells, PD-1 cells had a much lower MFI than – hi mice incubated for 5 h with anti PDL-1 or control antibody (Fig. cycling or noncycling PD-1 cells (Fig. S4), suggesting that cycling – hi + S7). Anti PDL-1 treatment mediated reduction in apoptosis PD-1 CD8 T cells exhibit poor survival. occurred primarily in the PD-1int subset and only moderate de- Because PD-1 has been reported to be transiently expressed in crease in apoptosis was noted in the PD-1hi subset. To further acute viral infections (27), increased PD-1 MFI during later address if anti–PDL-1 treatment was capable of rescuing pre- stages of chronic Toxoplasmosis could be because of a genera- + – existing PD-1 expressing CD8 T cells in vivo, we adoptively tion of new PD-1 expressing effectors in response to parasite transferred sorted PD-1+CD8+ T cells from chronically infected recrudescence. To address this theory, we used a BrdU pulse- CD45.1 mice (week 5–6) to similarly infected CD45.2 mice and chase approach and then measured PD-1 expression on BrdU+ – – − + then treated recipients with anti PDL-1 antibody. Anti PDL-1 and Brdu CD8 T cells during the early-chronic and late-chronic treatment increased the absolute number of donor cells in both phases (Fig. S5). Dramatic increase in PD-1 MFI and frequency of spleen and brain (Fig. S8). However, although the recipient hi fi + + − PD-1 cells was noted only in BrdU-labeled fraction. This nding CD8 T-cell population (comprising of both PD-1 and PD-1 ) suggests that PD-1 up-regulation is a graded process and the hi + expanded sixfold at the effector site (vis-à-vis isotype-treated), majority of PD-1 CD8 T cells at week 7 postinfection are not the donor population underwent over 20-fold expansion. This new effector cells generated in response to parasite recrudescence. fi – hi + nding suggests that anti PDL-1 treatment preferentially rescues Considering that a majority of PD-1 CD8 T cells during the preexisting PD-1–expressing cells. late-chronic phase are not newly generated effectors, we wanted to address if the control of infection during the early or later phases of Blockade of the PD-1–PDL-1 Pathway Controls Toxoplasma Recrudescence. infection would result in reduced CD8 exhaustion. To evaluate Finally, we wanted to address if this augmented CD8+ T-cell re- this, we treated mice with sulfamethoxazole and trimethoprim at sponse was able to control recrudescence by examining the gene various time points postinfection, as show in Fig. S6 (19). Because expression of Toxoplasma parasites in brains of anti–PDL-1 or reduced antigen burden in drug-treated mice can result in in- control antibody treated mice. Anti–PDL-1 treated mice revealed creased naive T-cell numbers, which can potentially confound more of a bradyzoite-specific than tachyzoite-specificgeneex- interpretation of data, we analyzed brain, a tissue known to recruit pression (Fig. 4A). Moreover, anti–PDL-1 treated mice exhibited CD8+ T cells in a strictly antigen-specific manner (30). Only early a much lower frequency of Toxoplasma-infected leukocytes in initiation of drug treatment resulted in decreased PD-1 MFI on brain and blood than control group (Fig. 4B). As previously stated, CD8+ T cells during the late-chronic phase (Fig. S6). Moreover, because encysted parasites can persist for life in immunocompe- CD8+ T cells from these mice exhibited a higher proportion of tent host brains without causing pathology, we examined parasite − IFN-γ+GranzymeB and IFN-γ+GranzymeB+ CD8+ T cells (Fig. genomic DNA in blood (6). Detection of parasite DNA in blood − S6). However, IFN-γ GranzymeB+ CD8+ T cells underwent by PCR has been used to diagnose cerebral toxoplasmosis in a moderate decrease in these animals (Fig. S6). This finding may patents with AIDS (36). As shown in Fig. 4C, parasite genomic

Bhadra et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 p=.0001 increased survival of anti–PDL-1 treated mice was CD8-dependent, A 9.0

6 the surviving mice were treated with anti-CD8 antibody. As shown 6.0 in Fig. 4E, CD8 depletion abrogated the survival of anti–PDL-1 + 10 x 3.0 treated animals. Taken together, the above data suggest that anti– p=.0002 +

5.0 PDL-1 treatment augments suboptimal CD8 T-cell response in 5 3.5 chronically infected mice, which prevents reactivation of infection

x 10 x leading to host survival. 2.0 p=.0006 1.1 – +

4 Anti PDL-1 Treatment Up-Regulates Eomes in CD8 T Cells from 0.6 Brain Liver Spleen Liver Brain Chronically Infected Mice.

x 10 x T-box factors, T-bet and Eomeso- Absolute Number of CD8 T cells T CD8 of Number Absolute 0.1 dermin, play a critical role in development, survival, and function Isotype Anti- of CD8+ T cells (37, 38). Studies from our laboratory suggest that Control PDL-1 anti–PDL-1 treatment dramatically augments expression of Isotype Control Anti-PDL-1 Eomes but not T-bet (unaffected vis-à-vis acutely infected mice) in + B lo int hi lo int hi cycling CD8 T cells (Fig. S9). Incidentally, the critical role of Eomes in mediating CD8+ T-cell responsiveness to IL-15 (38), + 14.8 15.1 17.9 22.9 a cytokine crucial for survival of memory as well as effector CD8 T cells, and Granzyme B (39–41) expression in T cells has been recently elucidated. Significantly, our data show that CD8+ T-cell

CD8 26.0 6.5 30.0 10.1 dysfunction affects Granzyme B expression more than IFN-γ production. Significance of Eomes expression in our current model will have to await further investigation. 28.6 3.2 25.2 4.4

Brain Liver Spleen Liver Brain Ki-67 Discussion + - Gated on CD8 + CD90.1 T cells CD8 T-cell exhaustion has been reported in several chronic C Isotype Control Anti-PDL-1 viral infections, like LCMV, HBV, HCV, HIV, and Simian Im- 5.2 4.8 4.1 9.1 munodeficiency Virus, which are characterized by high levels of persisting (42). This exhaustion is manifested by the gradual loss of CD8+ T-cell effector functions (cytokine pro- 18.6 29.2 duction, cytotoxicity, proliferation, and recall responses) and, in +

IFNγ 5.3 2.6 6.7 6.0 extreme situations, CD8 T cells can be physically deleted (43). In contrast, in infection models like murine cytomegalovirus,

Brain Spleen Brain characterized by low levels of persistent viremia, antigen-specific 23.0 33.5 T cells remain functional and respond vigorously to viral chal- Granzyme B lenge (42, 44). In models of reactivation like HSV that induce latent infection punctuated by periods of reactivation, functional + D 60 Infected + Isotype Control long-lived CD8 T-cell response is generated (45). Based on Infected + Anti-PDL-1 these findings, it has been suggested that CD8+ T-cell exhaustion 40 Naive occurs only in the presence of uncontrolled persistent viremia + 20 (42, 46–48). Our current report documents that CD8 T-cell

Percent Lysis Percent dysfunction is not restricted to viral infections alone but can be 0 10:1 20:1 40:1 extended to intracellular protozoan parasite, like T. gondii. Al- Effector:Target Ratio though CD8+ T-cell dysfunction observed in the present study is similar in certain facets to certain viral , in its de- – E Isotype Control pendence on PD-1 PDL-1 interaction, it discounts the conven- Anti-PDL-1 tional paradigm that CD8+ T-cell exhaustion occurs only in infectious disease models with uncontrolled persistent load. Moreover, notwithstanding the fact that the role of the

% of maximum PD-1–PDL-1 pathway has been addressed in chronic viral + PD-1 models of CD8 T-cell exhaustion, its significance in bacterial or parasitic models of CD8+ T-cell dysfunction has not been Fig. 3. Blockade of PD-1–PDL-1 pathway reinvigorates CD8+ T-cell response extensively investigated. in both lymphoid and nonlymphoid tissue in chronically infected mice. (A) Our studies demonstrate that despite a potent development of Chronically infected mice were treated with anti–PDL-1 for 2 wk and then + + CD8 T-cell immunity during the early stages of chronic infection, killed at week 7 postinfection. The absolute number of CD8 T cells was endogenous CD8+ T-cell response is unable to control parasite evaluated in the spleen, liver, and brain. (B) The frequency of proliferating reactivation at the later time points. This finding is in agreement CD8+ T cells was assessed by intracellular staining for Ki-67. (C) IFN-γ and Granzyme B production by CD8+ T cells was evaluated in splenocytes or brain with a previous study by Gazzinelli et al. (49), which demonstrated fi that parasite reactivation in B6 mice during late-chronic phases is cells from infected mice in the presence of TLA. (D) Antigen-speci c cyto- fi toxicity of splenic CD8+ T-cells was measured by 51Cr-release assay. (E) PD-1 associated with increased expression of tachyzoite-speci c genes expression on splenic CD8+ T cells from anti–PDL-1 or isotype control anti- in brain. Additionally, there is an increase in parasite levels in the + body-treated mice was evaluated by flow cytometry. The data represent periphery. A recent study has demonstrated the CD11b leuko- three experiments with at least four mice per group. cytes can act as shuttles for parasite migration during the acute infection phase (22). Hence, it would be interesting to investigate in future studies whether during parasite reactivation, CD11b+ leukocytes, which are preferentially parasitized during the late- DNA was only detectable in control antibody-treated and not in chronic phase, potentially serve a similar function and its role in drug or anti–PDL-1 treated blood. Next, we monitored long-term – mediating disease pathogenesis. Cumulatively, our results show survival of anti PDL-1 treated mice. Surprisingly, although 100% that long-term exposure to a persistent pathogen despite initial of control mice died by day 91 postinfection, 90% of anti–PDL-1 control of parasitemia can lead to CD8+ T-cell dysfunction and treated mice survived until the termination (day 125 postinfection) parasite reactivation. This CD8 exhaustion with concomitant PD- of the experiment (Fig. 4D). Furthermore, to demonstrate that 1 up-regulation is characterized by reduced effector-cell numbers,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1015298108 Bhadra et al. Downloaded by guest on September 27, 2021 A ENO-2 ENO-1 dysfunction. This finding suggests that high antigen burden or 1.5 p=0.012 2.0 p=0.038 inflammation (including anti-inflammatory cytokines) or both that 1.5 1.0 occurs during acute phase of infection may be primarily re- 1.0 0.5 sponsible for defective development of a long-lived robust CD8 0.5 response during the chronic phase (42, 50). Based on a recent 0.0 0.0 study by Chang et al. (32) demonstrating that T-cell fate im- Relative Expression Isotype Control Anti-PDL-1 printing can occur as early as the first cell division, it is hardly surprising that T-cell microenvironment (cytokine, antigen bur- Isotype Control Anti-PDL-1 B 26.7 0.2 41.1 0.1 den, and so forth) during the early phase of Toxoplasma infection can have such a profound effect during the later phases. Our findings are further supported by a study in an LCMV model 16.0 4.5 11.3 0.3 25.2 0.0 where antigen burden has been shown to be the key determinant CD8 for development of CD8 dysfunction (42). However, any control fl

Blood Brain Blood of infection or antigen burden can potentially affect in ammation. 36.5 3.9 T. gondii Hence, in future studies it will be critical to discriminate the dif- ferential role of inflammatory milieu and antigen burden in me- 30 diating CD8 dysfunction. C Recent reports have identified role of IL-10 in mediating + 20 dysfunctional CD8 T-cell response during LCMV infection (51). However, anti-IL10R treatment of mice carrying chronic 10 Toxoplasma infection led to their increased mortality because of fl fi 0 a strong in ammatory response (52). This nding suggests that

Parasite DNA levels Parasite DNA any strategy for preventing Toxoplasma reactivation must involve

per 400ng of whole blood fl

PDL-1

Anti- Control

Isotype an appropriate balance between enhanced proin ammatory

Treated Treated

Drug Treatment Pre mediators without excessive suppression of anti-inflammatory Week 5 Week 7 factors. Interestingly, anti–PDL-1 treatment not only augmented the CD8+ T-cell response but also prevented mortality in Anti-PDL-1 treatment D chronically infected mice by controlling parasite reactivation. – + How blockade of the PD-1 PDL-1 pathway modulates CD8 IMMUNOLOGY 0 35 49 125 T-cell response at the molecular level is poorly understood in Days post-infection 100 infectious disease models. Recent studies have shown that dif- 80 ferential TCR signal strength can affect not only memory CD8 p<0.0001 programming but also development of a potent polyfunctional 60 CD8 response (53, 54). Considering that PD-1 ligation has been 40 shown to inhibit TCR signaling via down-regulation of ZAP-70 20 Anti-PDL-1 θ –

Percent Survival and PKC in vitro, it is possible that anti PDL-1 treatment Isotype Control 0 restores robust CD8 response during chronic Toxoplasma in- 0 28 56 84 125 Days post-infection fection via increased TCR sensitivity (55). Although the mech-

anistic basis of CD8 dysfunction and PD-1 up-regulation need Anti-CD8 Anti-PDL-1 to be investigated more thoroughly in future studies, the data E treatment treatment presented in this article provide significant insights into the role

of PD-1–PDL-1 pathway in mediating CD8+ T-cell dysfunction 126 136 146 150 Days post-infection and concomitant parasite reactivation. These results are espe- 100 fi Anti-PDL-1 + cially signi cant in view of a minimal role played by CTLA-4, 80 Isotype Control a related inhibitory molecule in regulating T-cell response during 60 Anti-PDL-1 + Anti-CD8 TE (56). Our findings have important implications in the use 40 of PD-1–PDL-1 blockade in combination with drug regimen 20 p=.0067 for controlling TE in immunocompromised patients, as well as Percent Survival 0 126 136 150 use of similar therapy in other dysfunction-induced reactivation- Days post-infection type diseases. – Fig. 4. Anti PDL-1 treatment controls parasite reactivation and prevents Materials and Methods mortality in chronically infected mice. (A) Relative expression of ENO-1 and ENO-2 was computed for each sample. Transcript levels at day 10 post- Mice, Parasites, and in Vivo Antibody Treatment. Female C57BL/6, CD45.1, and infection was taken as 1. (B) T. gondii-infected cells in the brain and blood CD90.1 mice were purchased from the National Cancer Institute or the leukocyte-gated samples (n = 5) were assayed by flow cytometry. (C) Parasite Jackson Laboratory. Animal studies were carried out in agreement with In- genomic DNA level was computed in whole blood before initiation of stitutional Animal Care and Use Committee approved guidelines. Unless treatment (week 5 postinfection) or 2 wk after initiation of treatment (week otherwise mentioned, T. gondii cysts of ME49 strain were used for i.g. in- 7 postinfection) (n =4–5). (D) Anti–PDL-1 antibody was injected every third fection throughout. Some mice were injected in vivo with the following anti- day, starting at week 5 postinfection (n = 10). To minimize the development bodies i.p.: anti-PDL-1 (MIH5) or anti-CD8 (2.43) or isotype-matched control of an allogeneic response, the antibody was administered with 2-wk inter- antibodies. ruptions until termination of the experiment. (E) Surviving anti–PDL-1 treated mice from the previous experiment were administered anti–PDL-1 Lymphocyte Isolation, Cell Surface Staining, and Intracellular Staining. Single along with anti CD8 (n = 4) or anti–PDL-1 and control antibody (n = 4) every cell suspension was prepared from spleen, liver, blood, and brain using 5 third day unitl the conclusion of the experiment. Survival was monitored on standard protocol. For cytokine detection, 5 × 10 naïve congenic splenocytes a daily basis. Data represent two experiments. was mixed with equal number of brain or splenic cells from infected mice, and restimulation was similarly carried out in the presence of TLA.

Real-Time RT-PCR and PCR. From total infected mouse brain RNA, real-time RT- decreased polyfunctionality, increased apoptosis, and poor recall PCR was performed with primers for ENO-1, ENO-2, SAG-1, and BAG-1 on response and expansion. Preliminary studies using drug treatment Toxoplasma specific actin normalized samples. Transcript levels relative to suggest that control of infection during the early but not later day 10 post infection (day 10 post infection relative transcript level = 1.0) phases is critical for minimizing PD-1 up-regulation and CD8 were calculated according to the Pfaffl method of quantitation (57). Parasite

Bhadra et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 DNA quantitation (fg of DNA) was calculated after real-time PCR by com- Additional details and techniques are provided in SI Materials and paring Ct values for the B1 gene in experimental samples to a serial dilution Methods. of genomic parasite DNA equivalents as previously described (26). ACKNOWLEDGMENTS. We thank M. Azuma for providing MIH5 hybridoma. Statistical Analysis. Statistical analysis was performed used Student’s t test or This work was supported by National Institutes of Health Grant AI-33325 Log-rank test with P < 0.05 taken as statistically significant. (to I.A.K.).

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