Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection

Abstract

Infection of erythrocytes with Plasmodium species induces clinical malaria. Parasite-specific CD4(+) T cells correlate with lower parasite burdens and severity of human malaria and are needed to control blood-stage infection in mice. However, the characteristics of CD4(+) T cells that determine protection or parasite persistence remain unknown. Here we show that infection of humans with Plasmodium falciparum resulted in higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction. In vivo blockade of the PD-1 ligand PD-L1 and the inhibitory receptor LAG-3 restored CD4(+) T cell function, amplified the number of follicular helper T cells and germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, and proper function can be restored by inhibitory therapies to enhance parasite control.

Figure 1. Human and rodent malaria induce specific phenotypic and functional characteristics of CD4⁺ T cell exhaustion

(a) PD-1 expression by CD4⁺ T cells from Malian children before the malaria season (Before Malaria) and seven days after symptomatic P. falciparum infection (After Malaria). The non-parametric Mann-Whitney test was used to compare continuous variables between groups. (b) Resolution of P. yoelii (Py) blood-stage infection in rodents requires CD4⁺ T cells and antibody secreting B cells. Survival curves of Py pRBC infected wild-type C57BL/6 mice, wild-type mice depleted of CD4⁺ or CD8⁺ T cells on day 10, or C57BL/6 Aicda^–/– μs^–/– mice. Data represent 2 independent experiments with 5 mice/group. (c) Upregulation of CD49d and CD11a identifies Plasmodium-specific, infection-induced CD4⁺ T cells. Longitudinal analyses of PBL before (naïve) and 7 days following (Py pRBC) challenge. Data represent 4 independent experiments. (d) Prolonged Py blood-stage infection results in sustained CD4⁺ T cell proliferation. Histograms show the fraction of CD49d^hiCD11a^hi Plasmodium-specific (open) or CD49d^loCD11a^lo naïve (filled) CD4⁺ T cells that have incorporated BrdU following a day 4-8 pulse (top panel) or day 27-30 pulse (bottom panel). Data represent 3 independent experiments with 3 mice/group. (e) Chronic virus (LCMV cl13) and prolonged Plasmodium blood-stage infection (Py pRBC), but not acute virus infection (LCMV Arm) induce T cell inhibitory receptors PD-1 and LAG-3 at day 31 on splenic, pathogen-specific (open) but not naïve (filled), CD4⁺ T cells. Data represent 3 independent experiments with 3-5 mice/group. (f) Pathogen-specific CD49d^hiCD11a^hi CD4⁺ T cells from Py infected mice exhibit dysfunctional IFN-γ, TNF and IL-2 production in response to PMA/ionomycin stimulation. Data (mean±s.d.) are from 4-5 mice/group and are representative of 3 independent experiments. Statistics in (f) were determined by two-tailed, unpaired student's t-test.

Figure 2. Truncation of P. yoelii blood-stage infection with chloroquine reverses CD4⁺ T cell exhaustion

Groups of C57BL/6 mice were infected with 10⁵ P. yoelii pRBC and subsequently treated with PBS or 80 mg/kg chloroquine/PBS on day 8 and 9 p.i. Spleens were harvested from mice on day 8 (a), or day 24 p.i. (b) and cells were examined for the expression of the indicated cell surface markers or for the functional production of IFN-γ following ex vivo stimulation with PMA-ionomycin (PMA–iono). Numbers in histograms refer to frequency of CD49d^hiCD11a^hi cells expressing the indicated marker or IFN-γ. Numbers in parentheses show mean fluorescence intensity of T cell inhibitory receptor staining. Data in a,b are representative of 2 independent experiments with 5 mice/group.

Figure 3. Therapeutic in vivo blockade of PD-1 and LAG-3 in mice improves the anti-Plasmodial CD4⁺ T cell response and accelerates parasite clearance

(a) Starting on day 14 after infection with 10⁵ Py pRBC, C57BL/6 mice were treated with 200 μg each of anti-PD-L1 and anti-LAG-3, or control rIgG, every 3 days and monitored for parasite burden every two days. Data (mean±s.d.) are from 5 mice/group and are representative of 4 independent experiments. (b) Mice were infected and treated as in (a) and Plasmodium-specific (CD49d^hiCD11a^hi) splenic CD4⁺ T cells were enumerated on day 21 p.i. (c) Representative plots showing PMA–iono-induced cytokine expression by CD49d^hiCD11a^hi CD4⁺ T cells from rIgG- and inhibitory receptor blockade-treated mice analyzed on day 21p.i. (d) Summary cytokine expression by CD49d^hiCD11a^hi CD4⁺ T cells from rIgG- and inhibitory receptor blockade-treated mice. Data (Mean±s.e.m.) in (b) and (d) derive from 2 independent experiments with 3-5 mice/group. Statistics in (a), (d) and (d) were determined by two-tailed, unpaired student's t-test. (*=P<0.05, **P<0.01).

Figure 4. Therapeutic T cell inhibitory blockade accelerates parasite clearance in genetically diverse backgrounds and prevents chronic Plasmodium infection

(a) Female Swiss Webster mice (n=10/group) were infected with 10⁵ P. yoelii parasitized red blood cells and given 200 μg of anti-PD-L1 and anti-LAG-3, or control rIgG, every three days starting on day 14 p.i. Parasitemia was monitored in individual mice until day 24. Statistics were determined by two-tailed, unpaired student's t-test. Data (Mean±s.d.) are representative of 2 independent experiments. Asterisks indicate P < 0.005. (b-c) Therapeutic in vivo blockade of PD-1 and LAG-3 signaling results in sterilizing clearance of persistent, subpatent P. chabaudi chabaudi (Pcc) infection in the majority of mice. (b) C57BL/6 mice were infected with 10⁴ Pcc blood-stage parasites and given 200 μg of anti-PD-L1 and anti-LAG-3 every three days from days 14 to 32. Parasitemia was monitored on the indicated days. Data (Mean±s.d.) are from 5 mice/group. On day 40 post-challenge, 100 μl of whole blood was collected via cardiac puncture from each donor mouse, diluted 1:2 in saline and injected intravenously into new naïve recipient mice. Recipient mice were monitored for the development of patent Pcc parasitemia from day 2 to day 20 post-transfer. (c) Frequency of patent infection and quantification of parasite burden on day 9 post-transfer in recipient mice are shown. For b-c, the parasitemia limit of detection (L.O.D.) was 0.02%. Data in b-c are representative of 2 independent experiments.

Figure 5. Therapeutic in vivo blockade of PD-L1 and LAG-3 in mice enhances T_FH CD4⁺ T cell and plasmablast differentiation during clinical malaria

(a) Starting on day 14 after Py pRBC infection, groups of C57BL/6 mice were treated with 200 μg each of anti-PD-L1 and anti-LAG-3, or control rIgG, every 3 days. On day 21 p.i., splenic CD49d^hiCD11a^hiCXCR5^hiCD150^lo CD4⁺ T follicular helper (Tfh) cells were enumerated. (b) Mice were infected and treated as in (a) and CD19^hi/intB220^hi/int B cells were analyzed for early plasma cell differentiation (evidenced by coordinate downregulation of IgD and upregulation of CD138 prior to loss of CD19 and B220 expression). (c) Summary data (Mean±s.d.) showing total numbers of T_FH and pre-plasma cells in rIgG- and inhibitory receptor blockade-treated mice 21 days p.i. Statistics in (c) were determined by two-tailed, unpaired student's t-test. Data in a-c are representative of 3 independent experiments with 5 mice/group.

Figure 6. Enhanced germinal center B cell reaction, class switch recombination and functionally protective anti-Plasmodial antibody secretion following therapeutic PDL1 and LAG-3 blockade during clinical malaria

(a) C57BL/6 mice were infected with Py pRBC and subsequently treated with blocking antibodies as described for Figure 2. On day 21 p.i., total CD19⁺B220⁺ B cells were enumerated. Data (Mean±s.d.) represent 3 independent experiments with 3 mice/group. (b) Representative dot plots showing enhanced splenic germinal center (B220^hi/intPNA^hi) B cell responses on day 21 p.i. in mice that received anti-PD-L1 and anti-LAG-3 treatment from days 14 to 20 p.i. Histograms show the frequency of class-switching (IgM^lo and IgG2b^hi) in germinal center (PNA^hi) and non-germinal center (PNA^lo) B cells. (c) Summary data (Mean±s.d.) show total numbers of germinal center and class-switched B cells in rIgG- versus anti-PD-L1 and anti-LAG-3 treated mice and are representative of 3 independent experiments with 3 mice/group. Statistics in (a) and (c) were determined by 2-tailed unpaired student's t-test. (d) Sera from Py infected mice that were subsequently treated with rIgG or anti-PDL1 and anti-LAG-3 from days 14 to 32 were collected on day 41 p.i. Total MSP-1₁₉-specific IgG antibodies were detected as described in Methods. Data (Mean±s.d.) are expressed as average endpoint titers with absorbance readings below 0.2 (A₄₀₅) and are representative of 2 experiments with 4 mice/group. (e) Parasite burdens and clearance kinetics following Py pRBC challenge of naïve mice receiving passive transfer of 150 μl of serum from donor mice subjected to the indicated treatments during Py malaria. Donor serum was obtained 41 days after initial Py pRBC challenge (2 weeks after cessation of blockade therapy). Data (Mean±s.d.) in (e) were analyzed by One-way ANOVA with Tukey's post-test of multiple comparisons and are representative of 2 independent experiments with 4 mice/group (*P<0.05; **P<0.01; ***P<0.001 for rIgG versus α-PD-L1+α-LAG-3).