PD-L1, TIM-3, and CTLA-4 Blockade Fails To Promote Resistance to Secondary Infection with Virulent Strains of Toxoplasma gondii

KEYWORDS: atypical strains, CD4 T cells, CD8 T cells, CTLA-4, checkpoint blockade, PD-1, T cell exhaustion, TIM-3, Toxoplasma gondii, vaccine

INTRODUCTION

T cell exhaustion is a state of cellular hyporesponsiveness that occurs in response to continued antigen stimulation or inflammation, wherein T cells produce fewer cytokines and cytotoxic molecules, lower expression levels of activating receptors, and increased expression levels of inhibitory receptors (1). T cell exhaustion was first characterized in chronic viral infection models (2,–4) but is now widely studied in cancer (5,–8), bacterial infection (9, 10), and parasitic infection models (11,–13) and is in part a programmed response to limit immune pathology in these settings. The molecular signature of CD8 and CD4 T cell exhaustion has been thoroughly defined, and several markers have been identified to distinguish these hyporesponsive states (14,–16). Several key markers that label CD8 or CD4 T cells as exhausted have been identified: (i) high expression levels of the inhibitory receptors PD-1, LAG-3, TIM-3, CTLA-4, 2B4, BTLA, and CD160; (ii) lowered expression levels of the costimulatory receptors 4-1bb, ICOS, and OX40; and (iii) differential expression of the transcription factors Tbet, Eomes, BLIMP-1, and others (17,–19). Exhausted T cells may carry most, or a portion, of these markers, and marker expression varies with the disease model, severity of disease, and cell type. Treatment with neutralizing antibodies that target inhibitory receptors, or “checkpoint blockade,” has proven effective in reversing disease severity in a variety of mouse models for chronic viral infection and cancer (20, 21). Importantly, these observations have translated to high response rates, tumor regression, and even survival of late-stage melanoma patients (22,–25). Therefore, checkpoint blockade is predicted to have a major impact on the treatment of human infectious disease (26, 27), but for which category of pathogen (bacterial, fungal, protozoan, helminth, or viral), which stage of infection (acute, chronic, or secondary), and which checkpoint blockade strategies should be used are less clear.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that infects nearly all warm-blooded vertebrates and exhibits a great deal of genetic diversity, especially among “atypical” South American strains (28,–31). T. gondii strains differ in virulence in mice, with type I and most atypical strains being virulent and type II and type III strains being relatively less virulent (32,–35). By using these strains, the immune response to T. gondii can be examined under conditions of various infection intensities, a strategy that is commonly used to study T cell exhaustion in the lymphocytic choriomeningitis virus (LCMV) system. During the initial phase of infection, host control of T. gondii requires both innate and adaptive immune cells that make gamma interferon (IFN-γ) (36). Despite immune pressure, T. gondii rapidly disseminates to distal tissues (37) to chronically infect for the lifetime of the host. Both CD4 and CD8 T cells play pivotal roles in preventing reactivation of the chronic form of infection and in preventing toxoplasmic encephalitis (38,–42). In this context, T cell exhaustion is a critical component of disease progression (43). Chronic infection with the intermediate-virulence type II ME49 strain will cause CD8 T cells to upregulate the inhibitory receptor PD-1 and exhibit diminished effector functions, including reduced IFN-γ and granzyme B (GzmB) production, in genetically susceptible C57BL/6 mice (13, 44). Bhadra et al. rescued exhausted CD8 T cells and parasite recrudescence following antibody blockade of PD-1 ligand (PD-L1) (13). They also observed a BLIMP-1-dependent CD4 T cell exhaustion program, with increased inhibitory receptor expression and decreased IFN-γ production during chronic T. gondii infection (45). These results underscore the importance of T cell exhaustion and the clinical potential of checkpoint inhibitors to resolve chronic infections, including T. gondii infection.

Can checkpoint blockade therapies be used to treat acute parasitic infections? In early studies on the scope and efficacy of anti-CTLA-4 therapy, it was clearly demonstrated to be beneficial in mouse models of acute visceral leishmaniasis (46) and hookworm infections (47). Furthermore, given the current difficulties in vaccine design for many parasitic pathogens, perhaps immunotherapy could be used as a second option to treat vaccinated individuals who fail to control parasitic infection. By correcting impaired memory T cell responses, immunotherapy could have a profound impact on such individuals. Importantly, immunotherapy would be blind to antigen, major histocompatibility complex (MHC) allele type, and vaccine regimen of the infected individual and could work on antibiotic-resistant parasites. In mouse models of T. gondii reinfection (“secondary infection” or “challenge”), vaccinated (48,–51) or chronically infected (52) mice are not susceptible to secondary infections with the highly virulent type I RH strain. Although naive mice fail to control infection with as few as one parasite of the type I strain, adoptive transfer of memory CD8 T cells to naive mice confers protection (50, 53). While primary infection with vaccine or avirulent T. gondii strains can induce protective immunity to many virulent T. gondii strains, this is not true for most atypical strains (52).

Here we hypothesized that susceptibility of C57BL/6 mice to secondary infection may be due to dysfunctional T cell responses caused by highly virulent T. gondii strains. Moreover, we tested whether neutralization of inhibitory receptors that promote T cell dysfunction could induce mouse survival following secondary infection. Although CD8 T cells expressed exhaustion markers and exhibited diminished IFN-γ responses during secondary infection with virulent T. gondii strains, mice were not protected from challenge with the atypical strain MAS or the type I GT1 strain when administered neutralization antibodies to CTLA-4, TIM-3, and/or PD-L1.

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RESULTS

To explore the role of T cell exhaustion during acute secondary infections with T. gondii, genetically susceptible C57BL/6 mice were first injected intraperitoneally (i.p.) with the avirulent type III CEP hxgprt⁻ strain, which forms a nonlethal chronic infection, and 35 to 45 days later, mice were then reinfected (i.e., “secondary infection” or “challenge”) with either the atypical strains MAS and GUY-DOS or the highly passaged laboratory type I strain RH (Fig. 1A). All three T. gondii strains cause a lethal primary infection in naive mice (34, 35, 52); however, chronically infected C57BL/6 mice survive secondary infection with RH but not the MAS or GUY-DOS strain (52). In this model, susceptibility to secondary infection correlates with increased parasite numbers, which can be observed by bioluminescence imaging of mice between days 5 and 12 of secondary infection with luciferase-expressing MAS compared to the RH strain (Fig. 1B and data not shown). Consistent with cellular requirements for immunity reported in vaccination studies (48,–50), depletion of CD8 but not CD4 T cells after primary infection abrogated protection against RH challenge (Fig. 1C). Moreover, depletion of CD8 but not CD4 T cells impaired the ability of mice to control chronic infection. Both lineages of T cells are required to prevent reactivation in mice chronically infected with an intermediate-virulence type II strain, ME49 (39, 54), and CD8 T cells are the primary effector T (T_E) cells responsible for cyst removal in this setting (41, 55). Overall, our model, which uses an avirulent type III strain to generate immunological memory, is consistent with the above-mentioned models for chronic infection- and vaccine-induced immunity and positions memory CD8 T cells as central players in host resistance to T. gondii.

 

 

FIG 1

Requirement for CD8 T cells in a mouse model of T. gondii secondary infection. (A) Schematic of the model used to assess T cell exhaustion during secondary infection with virulent strains that cause lethal (MAS, GUY-DOS, and GT1) and nonlethal (RH) outcomes. Average percent survivals from previous results (52) are indicated. (B) Representative bioluminescence imaging of individual C57BL/6 mice following secondary infection with the RH (1-1) and MAS (2C8) luciferase-expressing strains. Relative parasite burdens are depicted as a heat map, and the maximum and minimum values were set to 10⁵ and 3 × 10³ photons/s/cm²/sr, respectively. (C) Following chronic infection, mice were treated with depletion antibodies to either CD8 or CD4 or given an isotype control antibody for 1 week and then challenged with RH (RH 2°) or not challenged (chronic). The depletion regimen was continued until day 11. Cumulative survival rates from two separate experiments are plotted (n = 4 mice per group); P values were obtained by using the log rank Mantel-Cox test comparing depleted cohorts against similarly infected isotype control-treated groups (n = 2 mice per isotype-treated group).

The T cell populations analyzed for exhaustion makers were defined by the expression of CD62L (L-selectin) and KLRG1 (killer cell lectin-like receptor G1). CD62L is a cell adhesion molecule important for homing lymphocytes into the T cell zone of secondary lymphoid tissues and is expressed on naive T cells and central memory T (T_CM) cells (56). In response to T cell receptor (TCR) triggering by antigen, T_CM cells initially produce a very minimal repertoire of cytokines, namely, interleukin-2 (IL-2), but following differentiation into effector T cells, these cells also produce large amounts of cytokines and granzyme (57). Effector memory T (T_EM) cells are active, cytokine-producing cells that, like T_E cells, do not express CD62L; instead, they express inflammatory chemokine receptors that home these cells to the site of infection. KLRG1 is a transmembrane protein found on lymphocytes that marks highly activated, terminally differentiated T_E and T_EM cells following microbial infections, including T. gondii infection (58,–60). CD8 T cells expressing CD62L or KLRG1 have different requirements for IL-12 during differentiation and different abilities to make IFN-γ during T. gondii infection (59, 61). Using this staining approach, the percentages (Fig. 2A and ​andB)B) and numbers (Fig. 2C) of CD62L⁺ KLRG1⁻ (“CD62L⁺”), CD62L⁻ KLRG1⁻, and CD62L⁻ KLRG1⁺ (“KLRG1⁺”) CD8 T cells isolated from the peritoneal cavity significantly increased following chronic infection compared to those in naive animals. On day 5 of secondary infection with either the type I RH strain or the atypical strains MAS and GUY-DOS, the relative percentages and cell numbers of each population remained constant compared to those during chronic infection. A similar trend was also observed for CD4 T cell populations in the peritoneum (Fig. 2). Changes in splenic T cell responses were found to be minimal in this system, as discussed below. Thus, peritoneal T_E- or T_EM-like T cells expand at the site of initial infection, and their level of cellularity remains high following challenge.

 

FIG 2

Frequencies of effector T cell populations following chronic and secondary infections with various T. gondii strains. (A) Representative flow plots of CD8⁺ and CD4⁺ (CD3⁺ CD19⁻) peritoneal T cells and their expression of CD62L and KLRG1 from naive C57BL/6 mice, mice that were infected with the type III CEP hxgprt⁻ strain and allowed to progress to chronic infection for 35 to 45 days (Chronic), or chronically infected mice that were challenged with the indicated T. gondii strains and analyzed on day 5 of secondary infection. Numbers are the frequencies of cells that fall within the indicated gate. (B) Average frequencies ± standard deviations (SD) of the indicated cell populations, CD62L⁺ KLRG1⁻ (CD62L⁺), CD62L⁻ KLRG1⁻, or CD62L⁻ KLRG1⁺ (KLRG1⁺), among total CD4⁺ or CD8⁺ (CD3⁺ CD19⁻) T cells. Each dot represents the data from one mouse, and cumulative results from three to five experiments for CD4 T cell analysis and from six to eight experiments for CD8 T cell analysis are shown. (C) Same as for panel B, except that absolute cell numbers in the peritoneum were inferred from fluorescent bead recovery after peritoneal wash and counting by FACS analysis. Cumulative results from two separate experiments are plotted. P values were calculated with one-way ANOVA. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. The top bar compares the means for all groups; the bottom bar compares the means for all infected mice of the following groups: chronic (Chr), RH, MAS, and GUY-DOS (GD).

Cell surface expression levels of several inhibitory receptors and one costimulatory receptor associated with T cell exhaustion in the LCMV model (14, 16), PD-1, TIM-3, CTLA-4, and 4-1bb, respectively, were measured on the various CD8 T cell populations described in the legend of Fig. 2. Compared to CD8 T cells from chronically infected mice or mice challenged with RH, CD8 T cells from mice challenged with the atypical strains MAS and GUY-DOS exhibited increased expression levels of PD-1 and TIM-3, most noticeably on the CD62L⁻ KLRG1⁻ and KLRG1⁺ (T_EM-like) populations (Fig. 3A and ​andB).B). The costimulatory tumor necrosis factor (TNF) superfamily receptor 4-1bb also showed modest but significantly increased expression on CD62L⁻ KLRG1^+/− CD8 T cell populations following virulent compared to nonvirulent secondary or chronic infections. The NK cell receptor 2B4, associated with T cell survival during secondary LCMV infections (62), was upregulated on CD62L⁻ KLRG1^+/− CD8 T cell populations following chronic infection compared to those in naive mice but was not upregulated in response to virulent secondary infections with the MAS strain (not shown). Because CTLA-4 expression was only marginally detected on the surface of CD8 T cells in this model (not shown), likely due to rapid internalization following T cell activation (63), intracellular staining was implemented. Increased CTLA-4 protein expression was observed in CD8 T cells following lethal secondary compared to nonlethal infections; however, statistical significance was not reached due to individual mouse variability. Finally, because the degree of CD8 T cell hyporesponsiveness correlates with the coexpression of multiple inhibitory receptors (64), the frequency of CD8 T cell populations that coexpress PD-1 and TIM-3 or PD-1 and CTLA-4 was determined. Compared to nonlethal conditions, secondary infections with virulent strains significantly enhanced the coexpression of PD-1 and TIM-3 on each CD8 T cell population analyzed (Fig. 3C and ​andD).D). The coexpression of CTLA-4 and PD-1 also followed a similar trend; however, statistical significance between lethal and nonlethal infections was achieved only for CD62L⁺ CD8 T cells, likely reflecting individual mouse variability in CTLA-4 expression. In summary, the expression of several T cell exhaustion markers, PD-1, TIM-3, 4-1bb, and, to a lesser extent, CTLA-4, on peritoneal CD8 T cells correlates with host susceptibility to secondary infection with T. gondii.

 

 

 

FIG 3

Markers of T cell exhaustion are highly expressed on CD8 T cells following secondary infection with virulent strains of T. gondii. (A) Average frequencies ± SD of CD62L⁺ KLRG1⁻ (CD62L⁺), CD62L⁻ KLRG1⁻, or CD62L⁻ KLRG1⁺ (KLRG1⁺) CD8⁺ (CD3⁺ CD19⁻) peritoneal T cells that express the indicated exhaustion markers. Each dot represents data from one mouse on day 5 of secondary infection with RH, MAS, and GUY-DOS (GD) or without challenge (chronic infection [Chr]); cumulative results from three to seven experiments for PD-1, TIM-3, and 4-1bb and from two experiments for CTLA-4 are plotted. Significant P values (one-way ANOVA) are indicated for the means for all infected mice or mice given a secondary infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Representative histogram plots of surface PD-1, TIM-3, or intracellular (i.c.) CTLA-4 expression for peritoneal CD62L⁻ KLRG1⁻ or KLRG1⁺ CD8⁺ T cells. Numbers represent the frequencies of cells that fall within the indicated gate; numbers and histogram lines are color-coded to match the indicated T. gondii infections. (C) Same as for panel A, except that the average frequencies ± SD of PD-1- and TIM-3- or CTLA-4 (i.c.)-coexpressing cells among the indicated CD8 T cell populations is plotted. Cumulative results from two to three experiments are shown. (D) Representative flow plots of peritoneal KLRG1⁺ CD8⁺ T cells for the expression of TIM-3 and PD-1. Numbers represent the frequencies of cells that fall within the indicated gates.

The same exhaustion marker profile was evaluated for CD4 T cells in the peritoneum (Fig. 4). Like CD8 T cells, CD4 T cells exhibited increased expression levels of PD-1, TIM-3, and 4-1bb (data not shown) in response to lethal secondary compared to nonlethal infections (Fig. 4A and ​andB).B). Subtle differences were noted between CD4 and CD8 T cells, in which higher frequencies of PD-1 but lower frequencies of TIM-3 were observed for KLRG1⁺ CD4 T cells than for their CD8 T cell counterparts. The level of CTLA-4, an inhibitory receptor highly expressed on CD4 T cells during exhaustion in the LCMV model (16), was also elevated in response to lethal challenge, but statistical significance was not obtained due to individual mouse variation. Because CTLA-4 is constitutively expressed on regulatory T cells (65), the frequency and contribution of these cells to the overall CTLA-4 signal in CD4 T cells were quantified (Fig. 4C and ​andD).D). The frequency of FOXP3⁺ CD25⁺ cells among total CD4 T cells dropped from 5.9% in naive mice to 1% following chronic infection or challenge. Although 97 to 100% of FOXP3⁺ CD25⁺ peritoneal CD4 T cells are CTLA-4⁺ (not shown), these cells represent 5% of the total CTLA-4 signal among CD4 T cells following T. gondii secondary infection (Fig. 4D). Therefore, effector CD4 T cells represent the majority of CTLA-4-expressing cells in the peritoneum following T. gondii infection. Finally, PD-1 expression correlated significantly with TIM-3 on all CD4 T cell populations analyzed and with CTLA-4 among KLRG1⁺ CD4 T cells following lethal compared to nonlethal infections (Fig. 4E and ​andF).F). In summary, the expression of T cell exhaustion markers on peritoneal CD4 T cells also correlates with host susceptibility to secondary infection, thus representing a shared characteristic of both T cell lineages in response to virulent challenge with T. gondii.

 

FIG 4

Markers of T cell exhaustion are upregulated on CD4 T cells following secondary infection with virulent strains of T. gondii. (A) Average frequencies ± SD of CD62L⁺ KLRG1⁻ (CD62L⁺), CD62L⁻ KLRG1⁻, or CD62L⁻ KLRG1⁺ (KLRG1⁺) CD4⁺ (CD3⁺ CD19⁻) peritoneal T cells that express the indicated exhaustion marker. Each dot represents data from one mouse on day 5 of secondary infection with RH, MAS, and GUY-DOS (GD) or without challenge (chronic infection [Chr]), and cumulative results from three to four experiments are plotted. Significant P values (one-way ANOVA) are indicated for the means for all infected mice or mice given a secondary infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Representative histogram plots of PD-1, TIM-3, and intracellular (i.c.) CTLA-4 expression for peritoneal CD62L⁻ KLRG1⁻ or KLRG1⁺ CD4⁺ T cells. Numbers represent the frequencies of cells that fall within the indicated gate; numbers and histogram lines are color-coded to match the indicated T. gondii infections. (C) Representative flow plots depicting CD25⁺ and FOXP3⁺ (i.c.) staining of total CD4⁺ or CTLA-4⁺ CD4⁺ T cells from naive mice or from mice on day 5 of secondary infection with the MAS strain. Frequencies of cells that fall within the gates are shown. (D) Average frequencies ± SD of FOXP3⁺ CD25⁺ cells among total CD4⁺ T cells (top) or total CTLA4⁺ CD4⁺ T cells (bottom). Statistical analysis was performed as described above for panel A, and cumulative results are plotted from two experiments. (E) Same as for panel A, except that the average frequencies ± SD of PD-1- and TIM-3- or CTLA-4 (i.c.)-coexpressing cells among the indicated CD4⁺ T cell populations are plotted. Cumulative results from two to three experiments are shown. (F) Representative flow plots of peritoneal KLRG1⁺ CD4⁺ T cells for the expression of TIM-3 and PD-1. Frequencies of cells that fall within the gates are indicated.

To assess whether increased expression of exhaustion markers on T cells correlated with a diminished capacity to produce IFN-γ, the critical cytokine required for immunity to T. gondii (43), its expression was measured by in vitro recall and intracellular flow analyses. Peritoneal exudate cells (PECs) were harvested from chronically infected C57BL/6 mice or from mice on day 5 of secondary infection with the various parasite strains. PECs were then infected with T. gondii in vitro (“recall”) and evaluated 16 h later for intracellular IFN-γ expression (52, 59). Relative to challenge with the RH strain, CD8 T cells from mice challenged with atypical strains displayed a diminished capacity to produce IFN-γ (Fig. 5A and ​andB).B). The recall IFN-γ response was independent of the parasite strain used for the in vitro recall infection (RH, MAS, or GUY-DOS) (Fig. 5A and data not shown). In contrast to CD8 T cells, and despite expressing exhaustion markers (Fig. 4), CD4 T cell IFN-γ production was not impaired but rather increased following secondary infection, and this response was irrespective of the parasite strain used for secondary infection (Fig. 5B). In the spleen, very minimal IFN-γ responses (Fig. 5C and ​andD)D) or exhaustion marker profile differences for PD-1, TIM-3, or CTLA-4 were observed for splenic T cells following RH, MAS, and GUY-DOS secondary infections (Fig. 5E and ​andFF and data not shown). Compared to peritoneal CD8 T cells from animals vaccinated with a replication-deficient type I RH strain, splenic CD8 T cell IFN-γ responses are known to be greatly reduced after challenge (50). Lowered splenic T cell responses may therefore represent a general feature following i.p. priming with less-virulent T. gondii strains, as observed previously (52).

 

 

FIG 5

CD8 T cells produce less IFN-γ following challenge with virulent strains of T. gondii, while CD4 T cells are unimpaired. (A) PECs harvested from C57BL/6 mice on day 5 of secondary infection with the indicated T. gondii strains were infected in vitro (“recalled”) with either the RH or MAS strain or not infected in vitro (“no recall”), and 16 h later, intracellular staining for IFN-γ and flow analysis were performed. Representative flow plots depicting the frequencies of CD8⁺ (CD3⁺ CD19⁻) T cells that stain positive for IFN-γ within the indicated gate are shown. The IFN-γ response was independent of the strain type used in the in vitro recall assay (not shown for GUY-DOS). (B) Average frequencies ± SD of IFN-γ⁺ CD4⁺ or IFN-γ⁺ CD8⁺ (CD3⁺ CD19⁻) peritoneal T cells from chronically infected mice or from mice challenged with the indicated parasite strains. Each dot represents the data from one mouse following in vitro recall with the RH strain, and cumulative data from one (GUY-DOS) to three experiments (all other infections) are plotted. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant (one-way ANOVA). (C) CD8⁺ T cells from the spleen were analyzed for IFN-γ expression from the same mice as for panel A. (D) Same as for panel B, except that the average frequencies ± SD of IFN-γ⁺ CD4⁺ or IFN-γ⁺ CD8⁺ (CD3⁺ CD19⁻) splenic T cells are shown. Cumulative data from two to three experiments are shown. (E and F) Average frequencies of PD-1⁺ CD62L⁻ KLRG1⁻ or CD62L⁻ KLRG1⁺ (KLRG1⁺) CD8⁺ (CD3⁺ CD19⁻) (E) or CD4⁺ (F) T cell populations. Each dot represents results from individual naive mice, chronically infected (Chr) mice, or mice challenged with RH, MAS, or GUY-DOS (GD) on day 5 of secondary infection. Cumulative results from two to three experiments are plotted, and statistics were calculated as described above for panel B.

PD-1—PD-L1 neutralization proved effective in promoting mouse survival during chronic infection with the intermediate-virulence type II ME49 T. gondii strain (13) but has not been tested during secondary challenge with virulent T. gondii strains. To test whether disease outcomes could be reversed following virulent challenge, several neutralizing antibodies were administered to block the pathways of inhibitory receptors most highly expressed on T cells in this system. Both the atypical strain MAS and the type I GT1 strain were used to understand the therapeutic efficacies of the various checkpoint blockade strategies. Like MAS and GUY-DOS, the GT1 strain also induces a lethal secondary infection in C57BL/6 mice (52) and induces the expression of PD-1 on CD62L⁻ T_EM-like CD8 T cells (Fig. 6E). Although the expression level of the PD-1 ligand PD-L1 was not directly measured, treatment with neutralizing antibodies against PD-L1 is routinely used to block PD-1—PD-L1 signaling (18, 20). PD-L1 blockade failed to rescue mice following challenge with the atypical strain MAS (Fig. 6A), which exhibited similar parasite burdens between treated and control cohorts (Fig. 6B and ​andC),C), and failed to rescue mice challenged with the GT1 strain (Fig. 6D). Combining PD-L1 blockade with anti-TIM-3 neutralizing antibodies promotes favorable disease outcomes in chronic LCMV infection (66) and in tumor models (7). However, neither TIM-3 blockade nor combination therapy with PD-L1 rescued mice following challenge with GT1 (Fig. 6D).

 

 

FIG 6

Neutralization with CTLA-4-, TIM-3-, and/or PD-L1-blocking antibodies fails to rescue C57BL/6 mice following challenge with virulent T. gondii strains. (A and D) Following secondary infection with either the virulent atypical strain MAS (A) or the type I strain GT1 (D), mice were injected i.p. with 200 μg of the following monoclonal antibodies on days 1, 3, 5, 7, 10, and 13 after challenge, and cumulative survival rates from one to two experiments are plotted: rat IgG2b isotype (GT1, n = 9; MAS, n = 11), anti-PD-L1 (GT1, n = 9; MAS, n = 10), anti-TIM-3 (GT1, n = 5), anti-CTLA-4 (GT1, n = 5; MAS, n = 4), anti-PD-L1 plus anti-TIM-3 (GT1, n = 9), and anti-PD-L1 plus anti-CTLA-4 (GT1, n = 5; MAS, n = 5). P values (log rank Mantel-Cox test) were calculated by comparing each therapeutic cohort against the control arm (isotype treated); all P values were >0.05 and considered not significant (n.s.). (B) Representative bioluminescence images of mice treated with anti-PD-L1 or the rat IgG2b isotype control on day 8 of secondary infection with the MAS-luciferase strain. (C) Same as for panel B, except that the average total body photon emissions (photons per second per square centimeter per steradian) ± SD are plotted. Each dot represents the measurement for an individual mouse, and cumulative data from two experiments are shown. The P value was >0.05 (one-way ANOVA), which was not significant. (E) Representative histogram plots from three separate experiments (n = 3 to 5 mice per experiment) of PD-1 expression on CD62L⁻ KLRG1⁻ or CD62L⁻ KLRG1⁺ (KLRG1⁺) CD8⁺ (CD3⁺ CD19⁻) peritoneal T cells during chronic infection or on day 5 of secondary infection with the type I RH or GT1 strain. Numbers represent the frequencies of cells that fall within the indicated gates and are color-coded to match the indicated T. gondii infections. (F) Representative flow plots of peritoneal CD8⁺ and CD4⁺ (CD3⁺ CD19⁻) T cells from mice treated with PD-L1 plus anti-CTLA-4 or with the isotype control on day 8 of secondary infection with the MAS strain. Intracellular detection of IFN-γ, IL-2, and GzmB was performed after in vitro recall infection, as described in the legend of Fig. 5; frequencies of cells that fall within each quadrant are shown. (G) Same as for panel F, except that the average frequencies of CD8 or CD4 T cells that express all three (triple), at least two (double), one (single), or none (negative) of the three immune mediators (IFN-γ, IL-2, and GzmB), are plotted. Results were obtained from two experiments (n = 3 to 4 mice per experiment), and a chi-squared test was used to compare the therapeutic versus control arms.

CTLA-4 attenuates T cell proliferation through competition for B7-1/2 ligands with the costimulatory receptor CD28 (67, 68) and by the recruitment of the PP2A phosphatase to the TCR-proximal signaling cascade, thereby deactivating Akt (69). CTLA-4 blockade is known to increase CD4 T cell IFN-γ responses in cancer patients (70). Moreover, since CTLA-4 neutralization converts memory CD8 T cells to effector T cells through Treg–CTLA-4-mediated suppression (71), and ctla4^−/− transgenic CD8 T cells demonstrated enhanced secondary but not primary proliferative responses to antigen stimulation (72), the role of CTLA-4 blockade was explored in this system. However, neither a single CTLA-4 blockade nor combination therapy with PD-L1 altered survival following challenge with either strain (Fig. 6A and ​andD).D). The capacity of peritoneal CD4 and CD8 T cells to produce IFN-γ, IL-2, and GzmB in the context of anti-PD-L1–anti-CTLA-4 combination therapy was explored. Following MAS challenge, modest but borderline significant (by chi-squared tests [Fig. 6G] and t tests [not shown]) increases in the frequencies of T cells that produce two or three of the measured immune mediators were observed for the treated compared to control cohorts (Fig. 6F and ​andG).G). Resistance to checkpoint blockade is known to be caused by systemic IFN-γ signaling (73, 74). Since IFN-γ is highly produced by many cell types and detected in the serum following secondary infection (data not shown), the sustained presence of IFN-γ may cause the minimal T cell reinvigoration observed in this system. Regardless, these results suggest that blockade of the inhibitory pathways studied here, which are commonly targeted to reverse disease outcomes in mouse models of cancer, LCMV, and chronic T. gondii infections, is not suitable to treat acute secondary infections with virulent T. gondii strains.

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DISCUSSION

The immune evasion mechanisms used by human parasitic pathogens, including T. gondii, are abundant and have contributed to the difficulty of parasitic disease prevention. Today, only one vaccine exists for any human parasitic pathogen, Plasmodium falciparum, and its efficacy is very low (25% efficacy; RTS,S) (75). Most vaccines fail to elicit long-lasting effector T cell responses (76), which are required to kill many parasitic pathogens (77, 78). Given the challenges with parasite vaccination and the estimated 3 billion people currently infected with parasites (79,–81), an exploration of inhibitory receptor pathways as targets in parasitic disease (12) or following vaccination warrants further attention. Since studies in the LCMV model have shown the usefulness of combining checkpoint blockade with vaccination to maximize protective immunity (82), and because anti-PD-L1 therapy prevents recrudescence during chronic T. gondii infection (13), we explored whether checkpoint blockade could cure virulent secondary infections with T. gondii.

Our data suggest that both CD4 and CD8 T cells express receptors associated with T cell exhaustion following challenge with virulent T. gondii strains and that CD8 T cells are hyporesponsive in this context. However, the individual impact of each of these T cell lineages and coreceptors that regulate immunity to reinfection with T. gondii is not yet resolved and has likely contributed to our inability to reverse disease outcomes. CD4 T cell help to activate naive CD8 T cells conventionally occurs through CD40-CD40L interactions on both antigen-presenting cells (APCs) (83) and CD40-expressing CD8 T cells (84). CD4 T cell help during primary infection is required to generate memory CD8 T cells that protect against T. gondii reinfection (85, 86). During prolonged antigen stimulation, memory CD8 T cells are more reliant on CD4 T cell help than are naive cells to control persistent LCMV infections (87), and CD40-CD40L interactions are likely involved (62). Work by Bhadra et al. suggests that the CD40-CD40L pathway plays a fundamental role in the rescue of exhausted CD8 T cells during chronic T. gondii infection (88). For example, following treatment with anti-PD-L1, CD40 was highly expressed on CD8 T cells, and CD8 T cell-intrinsic CD40 signaling played a major role in reinvigorating CD8 T cells during therapy (88). Moreover, the deletion of BLIMP-1 from CD4 T cells restored not only CD4 T cell function but also CD8 T cell function and control of T. gondii chronic infection (45). CD4 T cells may also be important in this system. Although CD4 T cells were not impaired in their ability to make IFN-γ, exhaustion markers were expressed on CD4 T cells following virulent secondary infections. Whether CD4 T cell helper functions fail to be propagated in this model of T. gondii reinfection is unknown. Further studies could be conducted to assess the role of CD40-CD40L signaling, other costimulatory pathways, and, more generally, CD4 T cell help during secondary challenge with virulent T. gondii strains. Costimulatory receptor agonists that mimic helper functions of CD4 T cells might be an avenue for therapeutic intervention in this system.

While PD-1 and TIM-3 were highly expressed on CD4 and CD8 T cells following virulent challenge with T. gondii, mice were refractory to therapeutics that target these inhibitory receptors. Previous studies in viral infection models have demonstrated that the state of progression of T cell exhaustion matters during rescue, and perhaps T cells are too exhausted to be rescued following virulent T. gondii infections. Exhausted T cells that are T-bet^hi and PD-1^int are more inclined to be rescued by inhibitory receptor blockade, while exhausted T cells that are EOMES^hi and PD-1^hi are more prone to die following blockade (17, 89). Furthermore, in mice chronically infected with LCMV, anti-PD-L1 therapy expanded a restricted population of exhausted CD8 T cells, defined by the expression of CXCR5, with properties similar to those of follicular helper T cells (90). Whether a therapeutically responsive population of exhausted T cells is present during T. gondii reinfection is unknown. Regardless, anti-CTLA-4 treatment, which has broad effects on recently activated T cells as well as memory T cells, did not rescue mice from virulent challenge. Furthermore, anti-PD-L1–anti-CTLA-4 combination therapy, which promotes superior melanoma clearance in mice (91) and heightened hepatitis C virus (HCV)-specific human CD8 T cell responses in vitro (92), again failed to rescue mice from secondary infections with virulent strains of T. gondii. While we have not explored the therapeutic effect of targeting all known exhaustion markers described for other systems, our data underscore the context-dependent effects of checkpoint blockade strategies on infection models (26). For example, PD-1 deficiency (93) or PD-1–PD-L1 antibody blockade (94, 95) prolongs mouse survival during bacterial sepsis but rapidly exacerbates disease during Mycobacterium tuberculosis infection (9, 10). Understanding the trade-off between enhanced resistance and immune pathology, as well as knowing the pathogen-specific mechanisms required for microbial killing, will be key for predicting the success of immunotherapeutic interventions for infectious disease.

Finally, while memory CD8 T cells are necessary for protection and are the only known memory population to adoptively transfer immunity to naive mice against T. gondii strains like RH (43, 50), there are likely additional requirements for immunity to more-virulent strains like GT1, MAS, and GUY-DOS. It is known that immunity cannot be achieved in B cell-deficient mice (96), suggesting a potential role for B cells and/or antibody production in resistance to highly virulent T. gondii strains. In summary, although the exhausted T cell phenotype correlates with increased parasite virulence, this likely represents an effect secondary to an underlying susceptibility factor present in C57BL/6 mice. Identifying this and additional requirements for host immunity to virulent strains of T. gondii is the subject of ongoing investigation.

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MATERIALS AND METHODS

Parasite strains and cells.

Human foreskin fibroblast (HFF) monolayers were grown at 37°C with 5% CO₂ in T-25 flasks for parasite passaging in HFF medium (Dulbecco's modified Eagle's medium [DMEM; Life Technologies] supplemented with 2 mM l-glutamine, 10% fetal bovine serum [FBS; Omega Scientific], 1% penicillin-streptomycin [Life Technologies], and 0.2% gentamicin [Life Technologies]). Toxoplasma gondii strains were passaged in HFFs in Toxo medium (4.5 g/liter d-glucose and l-glutamine in DMEM supplemented with 1% FBS and 1% penicillin-streptomycin). The following clonal strains were used (clonal types are indicated in parentheses): RH Δhxgprt (type I), RH Δhxgprt Δku80 (type I), RH GFP-cLUC (1-1) (type I), GT1 (type I), Pru (A7) GFP-fLUC Δhxgprt::HXGPRT (5-8B⁺) (type II), and CEP hxgprt⁻ (type III). The following atypical strains were used: MAS, MAS GFP-cLUC (2C8), and GUY-DOS.

Mice and ethics statements.

Six- to seven-week-old female C57BL/6J mice were purchased from Jackson Laboratory. For some experiments, CD45.1 congenic male and female C57BL/6 mice were used. Mouse work was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (97). All protocols were reviewed and approved by the UC Merced Institutional Animal Care and Use Committee. UC Merced has an Animal Welfare Assurance filed with OLAW (assurance number A4561-01), is registered with the USDA (registration number 93-R-0518), and the UC Merced Animal Care Program is AAALAC accredited (accreditation number 001318).

Primary infection and serotyping.

Parasite injections were prepared by scraping T-25 flasks containing vacuolated HFFs and sequential syringe lysis first through a 25-gauge needed and then through a 27-gauge needle. The parasites were spun at 400 rpm for 5 min, and the supernatant was transferred, followed by a spin at 1,700 rpm for 7 min. The parasites were washed with 10 ml phosphate-buffered saline (PBS), spun at 1,700 rpm for 7 min, and suspended in PBS. For chronic infections, mice were infected intraperitoneally (i.p.) with 10⁴ CEP hxgprt⁻ tachyzoites in 200 μl PBS. Parasite viability in the inoculum was determined by a plaque assay following i.p. infections. A total of 100 or 300 tachyzoites were plated in HFF monolayers grown in a 24-well plate, and 4 to 6 days later, plaques were counted by microscopy (4× objective).

At 30 to 35 days of chronic infection, 50 μl of blood was harvested from mice from the tail vein, collected in tubes containing 5 μl 0.5 M EDTA, and placed on ice. The blood was pelleted at 10,000 rpm for 5 min, and blood plasma was collected from the supernatant and stored at −80°C. To evaluate the seropositivity of the mice, HFFs were grown on coverslips and infected with green fluorescent protein (GFP)-expressing Pru (A7) or RH (1-1) overnight; 18 h later, cells were fixed with 3% formaldehyde in PBS, permeabilized with 3% bovine serum albumin–0.2 M Triton X-100–0.01% sodium azide, incubated with a 1:100 dilution of collected blood plasma for 2 h at room temperature, washed with PBS, and detected with Alexa Fluor 594-labeled secondary antibodies specific for mouse IgG (Life Technologies). Seropositive parasites were observed by immunofluorescence microscopy.

Secondary infections and bioluminescence imaging.

Seropositive mice were challenged with 5 × 10⁴ syringe-lysed parasites and euthanized on day 5 for most fluorescence-activated cell sorter (FACS) experiments or weighed every 3 to 4 days for survival experiments. Parasite viability for each strain was determined by a plaque assay following the completion of injections. The viability of the parasites ranged from 20% to 40% of the intended dose. In vivo bioluminescence imaging was performed on mice undergoing secondary infection with luciferase-expressing parasites as described previously (52).

Cell isolation, flow cytometry, and in vitro recall infections.

To isolate peritoneal exudate cells (PECs) by peritoneal lavage, 4 ml of PBS and 3 ml of air were injected into the peritoneal cavity with a 27-gauge needle. After shaking, the puncture was expanded with scissors, and the PEC wash was poured into a conical tube. PEC washes were filtered through a 70-μm cell strainer, pelleted, and washed with FACS buffer (PBS with 1% FBS) before staining. Spleens were dissected, crushed through 70-μm cell strainers, pelleted, suspended in ammonium chloride-potassium (ACK) red blood cell (RBC) lysis buffer for 5 min, quenched with medium containing 10% FBS, and washed with FACS buffer before staining.

For flow cytometry (flow) analysis, cells were further washed in FACS buffer prior to staining. All preparations were done on ice, and cells were blocked in FACS buffer containing Fc block anti-CD16/32 (2.4G2; BD Biosciences), 5% normal hamster serum, and 5% normal rat serum (Jackson ImmunoResearch) prior to staining with fluorophore-conjugated monoclonal antibodies (MAbs). The following MAbs (1:100 staining dilutions) were purchased from eBioscience (Thermo Fisher Scientific) unless otherwise stated: anti-CD4-phycoerythrin (PE)-Cy7 (GK1.5); anti-CD4-fluorescein isothiocyanate (FITC) (clone RM4-5); anti-CD8α-FITC, -allophycocyanin (APC), or -BV510 (53-6.7); anti-CD3ε-eFluor 780 (17A2); anti-CD62L-eFluor 450 (MEL-14); anti-KLRG1-FITC (2F1); anti-PD-1–PE or –APC (J43); anti-TIM-3–PE (RMT3-23); anti-4-1bb (CD137)–PE (17B5); anti-CD25-APC (PC61.5); and anti-CD19-peridinin chlorophyll protein (PerCP)/Cy5.5 (ebio1D3). All nonfixed samples were stained with propidium iodide (PI) (Sigma) at a final concentration of 1 μg/ml. PI-positive (PI⁺) and CD19⁺ cells were excluded from the analysis.

For cell enumeration, PBS used for peritoneal lavage was spiked with 5 × 10⁴ BD Calibrite APC beads (BD Bioscience). Beads were identified by FACS analysis as side-scatter high (SSCA^hi), PI negative (PI^neg), and APC⁺. The fraction of recovered beads was calculated by taking the count of identified beads and dividing this value by the initial quantity of 5 × 10⁴ beads. Cell enumeration was done by taking the counts of cell populations determined by FACS analysis and dividing them by the fraction of recovered beads per sample.

For the intracellular detection of CTLA-4, cells stained for the surface proteins CD19, CD3ε, CD62L, KLRG1, CD4, CD8α, and PD-1 were then fixed with BD Cytofix/Cytoperm and permeabilized with BD Perm/Wash solution (BD Pharmingen) according to the manufacturer's suggestions. Cells were stained with anti-CTLA-4 (CD152)–PE (UC10-4F10-11) (BD Biosciences) in Perm/Wash solution overnight, washed once with BD Perm/Wash solution and once in FACS buffer, and then analyzed by flow analysis. For the detection of FOXP3, cells were stained for surface proteins CD25, CD3ε, CD4, and CD19 and fixed by using a FoxP3/transcription factor fixation/permeabilization kit (eBioscience) for 45 min at room temperature. Fixed cells were washed and incubated in 1× permeabilization buffer for 10 min prior to intracellular staining with FOXP3-FITC (MF-14; eBioscience) and CTLA-4–PE (UC10-4F10-11) in 1× permeabilization buffer for 45 min and washed once with permeabilization buffer and once with FACS buffer prior to flow analysis.

For in vitro recall experiments, splenocytes and PECs were washed and plated in T cell medium (10% FBS in RPMI 1640 with GlutaMAX, antibiotics, 10 mM HEPES, 1 mM sodium pyruvate [Life Technologies], and 1.75 μl β-mercaptoethanol per 500 ml [MP Biomedicals]) at a concentration of 6 × 10⁵ cells per well (96-well plate). Cells were infected with the type I RH, MAS, or GUY-DOS strain at an MOI (multiplicity of infection) of 0.2 for 18 h, and 3 μg/ml brefeldin A (eBioscience) was added for the last 5 h of infection. Ninety-six-well plates were placed on ice, and cells were harvested by pipetting, washed with FACS buffer, blocked, and stained for the surface markers CD4, CD8α, CD3ε, and CD19. Next, cells were fixed with BD Cytofix/Cytoperm and permeabilized with BD Perm/Wash solution (BD Pharmingen) according to the manufacturer's suggestions. Cells were then stained with anti-IFN-γ–PE (XMG1.2), -granzyme B-FITC (GB11), and -IL-2–APC (JES6-5H4) on ice for 1 h or overnight for some experiments. Cells were then washed once with BD Perm/Wash solution and once in FACS buffer and analyzed by FACS. All data were acquired on an LSRII flow cytometer and analyzed with FlowJo software (TreeStar) to compensate and generate flow plots.

Neutralization and T cell depletions.

Mice were injected i.p. with 200 μg of the blocking antibodies anti-PD-L1 (10F.9G2), anti-TIM-3 (RMT3-23), and anti-CTLA-4 (9H10) or the rat IgG2b anti-keyhole limpet hemocyanin (KLH) (LTF-2) isotype control antibody, all of which were purchased from BioXCell, on days 1, 3, 5, 7, 10, and 13 after challenge with either the MAS or GT1 strain. Antibody injections were prepared in 200 μl of sterile PBS. Mouse survival and weight were monitored for 27 days. For T cell depletions, chronically infected mice were treated with 400 μg i.p. of anti-CD4 (GK1.5) or anti-CD8a (clone 2.43) on days −7 and −3, before challenge, and on days 3, 7, and 11 after challenge. The efficacy of depletion was determined by flow analysis of peripheral blood lymphocytes (PBLs) on days −1 and 8 of challenge. In brief, 15 μl of whole blood was collected and washed, and red blood cells were lysed with ACK lysis buffer, washed with FACS buffer, blocked, and stained with CD4-FITC (clone RM4-5) and CD8a-APC (clone 53.6-7), as described above. CD4 T cell depletion was determined to be 99.9% and 99.3% effective while CD8 T cell depletion was determined to be 99.7% and 99.5% effective on days −1 and 8, respectively (not shown).

Statistics.

One-way analysis of variance (ANOVA) was used to calculate significant differences between means of groups, a log rank (Mantel-Cox) test was used to assess differences in mouse survival between therapeutic treatment and control arms, and a chi-squared test was used to test categorical differences in T cell responses between therapeutic treatment and control arms. Statistics were calculated by using GraphPad Prism software, and a P value of <0.05 was considered significant.

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ACKNOWLEDGMENTS

We thank Jeroen Saeij (UC Davis) for initial support of this project and helpful advice and David Gravano (UC Merced) for support with flow analysis. We thank Angel Kongsomboonvech for generating the schematic in Fig. 1.

K.D.C.J. is supported by a Hellman fellowship (Hellman's fund) and the NIH (R15AI131027). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

S.D.S., S.P.S., and K.D.C.J. conceptualized and designed experiments. S.D.S., S.P.S., K.M.V., K.K.H., and K.D.C.J. analyzed experiments. S.D.S., S.P.S., K.M.V., B.E.C., A.B.C., and K.D.C.J. performed experiments. S.D.S., S.P.S., and K.D.C.J. wrote the manuscript.

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