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

Transcription

1 Supplementary Information Therapeutic PD L1 and LAG 3 blockade rapidly clears established blood stage Plasmodium infection Noah S. Butler, Jacqueline Moebius, Lecia L. Pewe, Boubacar Traore, Ogobara K. Doumbo, Lorraine T. Tygrett, Thomas J. Waldschmidt, Peter D. Crompton and John T. Harty Nature Immunology doi:1.138/ni.218

2 FSC SSC FSC PD US Control 1.3 Before Malaria After Malaria Supplementary Figure 1. Representative flow cytometric dot plots and gating strategy for analyses of PD-1 expression on + T cells from a U.S. donor control or a single subject examined before malaria and after diagnosis of clinical malaria during an active period of Plasmodium falciparum transmission. Cryopreserved PBMC samples were thawed and examined for the expression of and PD-1 as described in Methods. Nature Immunology doi:1.138/ni.218

3 a LCMV (d8 p.i.) LM (d7 p.i.) c Splenic MNC.6 No stim d 2 nm peptide IFN-g + IFN-g SMARTA Endo d SMARTA (d7 LCMV) b d d 9d Donor x1 5 9d hi hi 2x1 5 9d lo lo 5.1 Recipients 1 5 Py prbc Challenge e Donor of circulating + T cells (%) d hi hi 9d lo lo Time (d) Supplementary Figure 2. Validity of the 9d hi hi surrogate activation marker approach to identify virus-, bacterium- or Plasmodium-specific effector and memory + T cells. (a) Epitope-specific effector + T cells from lymphocytic choriomeningitis virus (LCMV)- or Listeria monocytogenes (LM)-infected mice exclusively exhibit the 9d hi hi phenotype. (b) Reverse gating strategy demonstrating that only the 9d hi hi population of + T cells from LCMV-infected mice express the effector cytokine. (c) Adoptive transfer of LCMV-specific (GP 61 ), naive transgenic + T cells (SMARTA, ) to P. yoelii infected mice (5.2 + ) does not result in upregulation of 9d or on virus-specific SMARTA Tg + T cells, whereas >3% of the endogenous splenic + T cells in these mice respond to P. yoelii blood-stage infection. Right panel demonstrates that virus-specific SMARTA transgenic + T cells become 9d hi hi following LCMV infection. Experimental design (d) and data (e) demonstrating that following adoptive transfer of Plasmodium-specific (9d hi hi ) or naïve (9d lo lo ), sortpurified + T cells from a day 71 P. yoelii-immune mouse, only the 9d hi hi memory + T cells undergo secondary expansion in a new naïve host subsequently infected with blood-stage P. yoelii infection. Data in a-c, e are representative of 2-3 independent experiments with 3-5 mice/group. Nature Immunology doi:1.138/ni.218

4 a 2.7 b c Naive Py prbc Events (% of max) 1 PD-1 LAG-3 CD16 2B BrdU d9 d31 d hi LCMV Arm <APC-A> 69 LCMV cl <APC-A> 52 e Py prbc Cytokine+ of hi T cells (%) P=.8 Events (% of max) Marker P= LCMV Arm LCMV cl13 Py prbc P=.8 TNF IL-2 Supplementary Figure 3. Sustained proliferation of Plasmodium-specific + T cells is associated with their acquisition of phenotypic and functional characteristics of T exhaustion during clinical malaria. (a) Coordinate upregulation of and downregulation of α identifies high-magnitude, Plasmodium-specific, infection-induced + T cells. (b) Mice were pulsed with BrdU d4-8 (top panels) or d27-3 (bottom panels) following Py prbc challenge. Splenic + T cells were stained for BrdU incorporation on the indicated days. Histograms show the frequency of Plasmodium-specific hi α lo (open) or naïve lo α hi (filled) + T cells that have incorporated BrdU following each of the indicated 4 day pulses. (c) Chronic virus (LCMV cl13) and prolonged Plasmodium blood-stage infection (Py prbc), but not acute virus infection (LCMV Arm) results in sustained, high-magnitude + T cell responses ( hi α lo ) with cells expressing T cell inhibitory receptors PD-1, LAG-3, CD16 and 2B4. Thirty one days p.i., splenic hi α lo pathogen-specific (open) and lo α hi naïve (filled) + T cells were surface stained for the expression of the indicated T cell inhibitory receptors. Representative dot plots (d) and summary data (e) demonstrating significantly impaired cytokine expression by + T cells responding to chronic Plasmodium blood-stage infection. Cells were stimulated ex vivo with PMA-iono and intracellularly stained for expression of the indicated cytokines 31 days following infection with LCMV Armstrong, LCMV cl13 or Py prbc. Statistics in (e) were determined by two-tailed, unpaired student s t-test. Data in a-c represent 2-3 independent experiments with 4-5 mice/group. Data (Mean +/- SD) in e represent 2 independent experiments with 5 mice/group <APC-A> Nature Immunology doi:1.138/ni.218

5 a b c PBS PBS Chloroquine PBS Events (% of max) Events (% of max) PD-1 Marker PD-1 LAG-3 2B4 CD16 Marker 43 1 No Stim (56) (162) LAG-3 66 (193) (482) 2 (49) (82) PMA-Iono 1.2 d (622) (284) 19 (87) (113) d24 Chloroquine TNF IL-2 6. d24 Supplementary Figure 4. Truncation of P. yoelii blood-stage infection reverses phenotypic and functional characteristics of + T cell exhaustion. Mice were infected with 1 5 P. yoelii prbc and left untreated or treated with 8 mg/kg chloroquine on days 8 and 9 p.i. Spleens were harvested from groups of mice on day 8 (a), or day 24 p.i. (b) and cells were examined for the expression of the indicated cell surface markers (a-b) and for the functional production of cytokines (c) following ex vivo stimulation with PMA-ionomycin. Numbers in histograms refer to frequency of hi α lo cells (open histograms) expressing the indicated marker, relative to naive lo α lo cells (filled histograms). Numbers in parentheses show mean fluorescence intensity of T cell inhibitory receptor staining. Data in a-c are representative of 3 experiments with 3 mice/group. 2B4 and CD16 expression levels were not examined on d8. Nature Immunology doi:1.138/ni.218

6 c a hi α lo T cells/spleen (x1 6 ) hi α lo T cells/spleen (x1 5 ) P=.3 rigg α-pd-l1 + α-lag-3 P=.2 P=.3 b rigg α-pd-l1 + α-lag TNF-α No stim PMA-Iono.9 18 IL TNF + + IL-2 + Supplementary Figure 5. Therapeutic in vivo blockade of PD-L1 and LAG-3 in mice with established clinical malaria reduces parasite burden, accelerates clearance and numerically and functionally improves the anti-plasmodial + T cell response. (a) Groups of mice were challenged with 1 5 Py prbc. Staring on day 14 p.i., mice were treated with 2 μg each of anti- PD-L1 and anti-lag-3, or control rigg, every 3 days. Plasmodium-specific ( hi α lo ) splenic + T cells were enumerated with flow cytometry on day 21 p.i. (b) Representative dot plots showing PMA-iono-induced cytokine expression by hi α lo + T cells from riggversus anti-pd-l1 + anti-lag-3-treated mice. Cells were stimulated and subsequently stained for intracellular and TNF or IL-2. (c) Summary data (Mean +/- SD) of cytokine expression by hi α lo + T cells from rigg- and inhibitory receptor blockade-treated mice. Statistics in (a) and (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. Nature Immunology doi:1.138/ni.218

7 Parasitemia (%) rigg α-pd-l1 α-lag/3 α-pd-l1 + α-lag Ab Time (d) Supplementary Figure 6. Therapeutic administration of anti-lag-3 synergistically acts with anti-pd-l1 blockade to reduce parasite burden and enhance parasite clearance. Four groups of 5-8 female C57BL/6 mice were infected with 1 5 P. yoelii prbc. Thirteen days later, parasitemia was assessed in individual mice and groups were treated with the indicated T cell inhibitory receptor blockade therapies every 3 days from day 14 to 32 postchallenge as detailed in Methods. Parasitemia was monitored every two days for the duration of the experiment. Data were analyzed by One-way ANOVA followed by Tukey s post-test for multiple comparisons. P =.3, rigg vs. α-pd-l1; P =.1, rigg vs. anti-pd-l1 + anti- LAG-3. Other comparisons were not statistically significant. Data are representative of two independent experiments. Nature Immunology doi:1.138/ni.218

8 rigg α-pd-l1+α-lag-3 Supplementary Figure 7. Immunostaining of frozen spleen sections harvested from rigg- versus anti-pd-l1 and anti-lag-3 blockade-treated mice. Groups of mice were challenged with 15 Py prbc. Staring on day 14 p.i., mice were treated with 2 μg each of anti-pd-l1 and anti-lag-3, or control rigg, every 3 days. Twenty one days p.i., spleens from control rigg-treated and anti-pd-l1 + anti-lag-3-treated mice were excised and frozen sections were prepared for immunostaining as described in Methods. Red staining corresponds to PNA+ germinal centers, whereas green denotes IgM+ B cell follicles. Images (5x magnification) were captured and tiled using a LSM 51 Zeiss laser-scanning confocal microscope. Data are representative of 15 sections from 2 independent mice from each experimental group. Nature Immunology doi:1.138/ni.218