Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α
- NIHR Biomedical Research Centre, School of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom
- Institute for Life Sciences, University of Southampton, United Kingdom
- Africa Health Research Institute, South Africa
- Department of Tropical and Infectious Diseases, Institute of Primate Research, National Museums of Kenya, Kenya
- Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa
- TB Centre and Department of Clinical Research, London School of Hygiene and Tropical Medicine, United Kingdom
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, United Kingdom
- NIHR Biomedical Research Centre, School of Cancer Sciences, University of Southampton, United Kingdom
- The Francis Crick Institute, United Kingdom
- BioPhysics Group, Department of Mechanical Engineering, University College London, United Kingdom
- Department of Infectious Diseases, Imperial College London, United Kingdom
- Department of Infection and Immunity, University College London, United Kingdom
Figures
Figure 1 with 2 supplements
PD-1 is expressed in human TB granulomas.
(A) Analysis of PD-1 expression by T cells in the lung and peripheral circulation of thirty-five TB patients undergoing medically indicated lung resection. PD-1 shows a trend towards higher …
Figure 1—figure supplement 1
PD-1 expression on peripheral CD4+ and CD8+ T-cells is predominantly on CD103- and CD69-negative cells.
Significance was analyzed by one-tailed unpaired Mann-Whitney test.
Figure 1—figure supplement 2
PD-1 expressing cells are absent in the immediate region surrounding caseous necrosis in human TB granulomas.
Images from four different clinical samples are presented. Scale bar 100 μm.
Figure 2 with 2 supplements
The PD-1/PD-L1 axis is upregulated in the 3D TB granuloma system.
(A) Still images from time-lapse microscopy imaging demonstrating increasing cellular aggregation of PBMC around a focus of ultraviolet killed Mtb H37Rv in the 3D granuloma system at times 0, 12, 24 …
Figure 2—figure supplement 1
Hypoxia alone has no significant effect on expression of the PD-1/PD-L1/2 axis.
Gene expression PD-1 and its ligands in uninfected cells was compared between normoxic and hypoxic conditions in the microsphere model. RNA was extracted from uninfected PBMCs and relative …
Figure 2—figure supplement 2
Mtb growth is reduced in hypoxia, while host cell viability is unchanged.
(A) Mtb H37Rv ffluc+ growth in 1% hypoxia. Microspheres were decapsulated in a media containing 5 mM EDTA and 0.1% saponin at day 14. The lysate was centrifuged, re-suspended in 1 ml PBS, serially …
Figure 3 with 1 supplement
PD-1 pathway inhibition increases Mtb growth.
(A) Inhibition of PD-1 receptors by small chemical inhibitor one increases Mtb growth in a dose-response manner (1–1000 nM). Inhibitor concentration 1 nM (green), 10 nM (purple), 100 nM (blue) and 1 …
Figure 3—figure supplement 1
Small chemical inhibition of PD-1/PD-L1 interaction in 1% hypoxia measured at day 14 shows a dose-dependent increase in Mtb growth with PD-1 inhibition.
***p<0.0001 with one-way ANOVA with Dunnett’s multiple comparison test.
Figure 4 with 3 supplements
PD-1 inhibition increases secretion of multiple cytokines and growth factors.
PD-1/PD-L1 signalling was inhibited by Spartalizumab, a humanized IgG4 anti-PD1 monoclonal antibody, in Mtb-infected microspheres at 20 and 200µ/ml in normoxia. Supernatants were collected at day 14 …
Figure 4—figure supplement 1
Cytokine accumulation around microspheres after inhibition of PD-1/PD-L1 signalling with Spartalizumab, a humanized IgG4 anti-PD1 monoclonal antibody at 20 and 200 µg/ml in normoxia (N) and 1% hypoxia (H).
Supernatants were collected at day 14 and a Luminex 35-multiplex assay was performed. The experiment was performed twice with three replicates. Concentrations are in pg/ml. Labels in red correspond …
Figure 4—figure supplement 2
PD-1 inhibition increases secretion of multiple cytokines and growth factors in 1% hypoxia.
Supernatants were collected at day 14 and accumulation of cytokines and growth factors was analysed by Luminex 35-multiplex assay. Concentrations were normalized to secretion by Mtb infected …
Figure 4—figure supplement 3
Spartalizumab induces TNF-α secretion in uninfected and infected microspheres, which is neutralised by anti-TNF-α.
PBMCs which were uninfected or infected with Mtb were encapsulated in alginate-collagen matrix after pre- incubation in Spartalizumab at 200 μg/ml. The supernatant was collected to measure TNF-α …
Figure 5 with 4 supplements
Addition of TNF-α increases Mtb growth in microspheres in normoxia.
Recombinant human G-CSF, GM-CSF, IL-4, IL-6, IL-10, IL-12, TNF-α, IL-1RA, MIP-1α, MIP-1β or RANTES were added either individually (A and B) or in combination pools (C) to Mtb-infected microspheres …
Figure 5—figure supplement 1
Individual Mtb growth curves at ‘high’ cytokine concentration, five times the concentration measured in media after anti-PD-1 treatment.
Human recombinant G-CSF, GM-CSF, IL-4, IL-6, IL-10, IL-12, TNF-α, IL-1RA, MIP-1α, MIP-1β and RANTES were added to microspheres either individually or in combination pools to microspheres at five …
Figure 5—figure supplement 2
Anti-TNF-α neutralizing antibodies supress the Mtb growth following TNF-α from a different source (Anti-TNF-α from Sigma-Aldrich, UK).
Figure 5—figure supplement 3
TNF-α skews polarization of monocytes to macrophages with lower CD80 expression.
PBMCs were infected with Mtb H37Rv at MOI of 0.1 and encapsulated in alginate-collagen microspheres after overnight incubation. Microspheres were then incubated in complete RPMI (with L-Glutamine …
Figure 5—figure supplement 4
Hierarchical gating strategy used to identify lymphocyte and monocytic populations from decapsulated microspheres containing human peripheral blood monocular cells.
Single cells were decapsulated from microspheres in 5 mM EDTA, washed and processed for flow cytometry. First doublets were excluded from live cells, then cells were gated as CD3+ and CD3-. …
Figure 6
TNF-α is expressed in human TB granulomas and sputum TNF-α concentrations negatively correlate with circulating PD-1 expression.
(A) TNF-α is expressed within human lung TB granulomas, with greater immunoreactivity than control lung tissue at the excision margin of lung cancer (i and ii). Quantification of TNF-α …
Videos
Video 1
Cell migration over 48 hr around a central cluster of macrophages infected with UV-killed Mtb within a 3D alginate-collagen matrix.
Migration is seen in the first 24 hr, without aggregation, and then progressive granuloma formation occurs.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv | (From Ref: 20) | Used at multiple of infection of 0.1 | |
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv pMV306hsp+LuxAB+G13+CDE | (From Ref: 20) | Used at multiple of infection of 0.1 | |
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv pMV306hsp encoding the wild-type FFluc | (From Ref: 20) | Used at multiple of infection of 0.1 | |
| Biological sample (Antibodies) | ||||
| Antibody | anti-CD45-V500 Horizon (Clone no. HI30) | BD Biosciences | Cat.No.563792 | Monoclonal mouse antibody |
| Antibody | anti-CD3 Brilliant Violet 785 (Clone no. OKT3) | Biolegend | Cat.No.317330 | Monoclonal mouse antibody |
| Antibody | anti-CD3-PE (Clone no. HIT3a) | Biolegend | Cat.No.300308 | Monoclonal mouse antibody |
| Antibody | anti-CD4 Brilliant Ultra Violet 496 (Clone no. SK3) | BD Bioscience | Cat.No.612937 | Monoclonal mouse antibody |
| Antibody | anti-CD4-PerCP (Clone no. OKT4) | Biolegend | Cat.No.317432 | Monoclonal mouse antibody |
| Antibody | anti-CD8 Brilliant Violet 605 (Clone no. RPA-T8) | Biolegend | Cat.No.301040 | Monoclonal mouse antibody |
| Antibody | anti-CD8-APC (Clone no. SK1) | Biolegend | Cat.No.344722 | Monoclonal mouse antibody |
| Antibody | anti-CD103-APC (Clone no. Ber-ACT8) | BD Biosciences | Cat.No.563883 | Monoclonal mouse antibody |
| Antibody | anti-CD69 Brilliant Ultra Violet 395 (Clone no. FN50) | Biolegend | Cat.No.310902 | Monoclonal mouse antibody |
| Antibody | anti-HLA-DR-PerCP (Clone no. L243) | Biolegend | Cat.No.307628 | Monoclonal mouse antibody |
| Antibody | anti-CD279-BB515 (Clone no. EH12.1) | BD Biosciences | Cat.No.564494 | Monoclonal mouse antibody |
| Antibody | anti- CD-274-BB515 (Clone no. MIH1) | BD Biosciences | Cat.No.564554 | Monoclonal mouse antibody |
| Antibody | anti-PD-1 Brilliant Violet 421 (Clone no.EH12.1) | BD Biosciences | Cat.No.562516 | Monoclonal mouse antibody |
| Antibody | anti-CD11b-APC (Clone no. ICRF44) | Biolegend | Cat.No.301310 | Monoclonal mouse antibody |
| Antibody | anti-CD45-APC/Cy7 (Clone no. 2D1) | Biolegend | Cat.No.368516 | Monoclonal mouse antibody |
| Antibody | anti-CD14-AP/APC (Clone no. HCD14) | Biolegend | Cat.No.325608 | Monoclonal mouse antibody |
| Antibody | anti-True-Stain Monocyte Blocker | Biolegend | Cat.No.426102 | Monoclonal mouse antibody |
| Antibody | anti-CD4 (Clone no. M7310) | DAKO | Cat.No.M7310 | Monoclonal mouse antibody |
| Antibody | anti-CD8 (Clone no. M7103) | DAKO | Cat.No. M7103 | Monoclonal mouse antibody |
| Antibody | anti-PD1 (Clone no. ab5287) | Abcam | Cat.No.ab52587 | Monoclonal mouse antibody |
| Antibody | anti-TNF-α (Clone no. ab1793) | Abcam | Cat.No.ab1793 | Monoclonal mouse antibody |
| Antibody | Spartalizumab | Selleckchem | Cat.No.A2017 | 20 µg/ml and 200 µg/ml, monoclonal, mouse IgG4 |
| Antibody | IgG4 | Sino Biologicals | Cat.No.13505-HNAH | 20 µg/ml and 200 µg/ml,monoclonal, mouse IgG4 |
| Antibody | Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), | Thermo Fisher Scientific | Cat.No.16-4714-82 | 50 µg/ml, mouse monoclonal IgG2A |
| Antibody | Mouse IgG1 Negative Control, clone Ci4 | Merck Life Sciences | Cat.No.MABC002 | 51 µg/ml, mouse monoclonal IgG2A |
| Antibody | anti-TNF-α | Thermo Fisher Scientific | Cat.No.16-7348-81 | 52 µg/ml, mouse monoclonal IgG2A |
| Sequence-based reagent (Applied Biosystems TaqMan Gene Expression primers) | GAPDH | Thermo Fisher Scientific | #Hs02758991_g1 | |
| β2-Microbulin | Thermo Fisher Scientific | #Hs00608023_m1 | ||
| FNTA | Thermo Fisher Scientific | #Hs00357739_m1 | ||
| PDCD1 | Thermo Fisher Scientific | #Hs01550088_m1 | ||
| CD274 | Thermo Fisher Scientific | #Hs00204257_m1 | ||
| PDCD1LG2 | Thermo Fisher Scientific | #Hs00228839_m1 | ||
| HIF-1α | Thermo Fisher Scientific | #Hs00153153_m1 | ||
| Commercial assay or kit | CytoTox-Glo Cytotoxicity Assay | Promega | G9291 | Commercial assay or kit |
| Lactate Dehydrogenase Activity Assay Kit | Merck | 11 644 793 001 | Commercial assay or kit | |
| Cytokine and Chemokine 35-Plex Human ProcartaPlex Panel | Thermo Fisher Scientific | LHC6005M | Commercial assay or kit | |
| Chemical compound, drug | PD-1/PD-L1 Inhibitor 1 | Cambridge Biosciences, UK | #1675201-83-8 | Chemical compound |
| Recombinant Human G-CSF | ImmunoTools | Cat.No.11343115 | 1 ng/ml and 5 ng/ml | |
| Recombinant Human GM-CSF | ImmunoTools | Cat.No.11343125 | 0.25 ng/ml and 1.25 ng/ml | |
| Recombinant Human IL-1RA/IL1 F3 | ImmunoTools | Cat.No.11344876 | 1.25 ng/ml and 6.25 ng/ml | |
| Recombinant Human IL-10 | ImmunoTools | Cat.No.11340105 | 0.2 ng/ml and 1 ng/ml | |
| Recombinant Human IL-6 | ImmunoTools | Cat.No.11340066 | 10.0 ng/ml and 50.0 ng/ml | |
| Recombinant Human IL-12 | ImmunoTools | Cat.No.11349125 | 0.5 ng/ml and 2.5 ng/ml | |
| Recombinant Human TNF-α | ImmunoTools | Cat.No.11343017 | 0.3 ng/ml, 1.5 ng/ml,7.5 ng/ml and 15 ng/ml | |
| Recombinant Human IL-15 | ImmunoTools | Cat.No.11340155 | 0.5 ng/ml, 5 ng/ml and 50 ng/ml | |
| Recombinant Human IL-17A | ImmunoTools | Cat.No.11340176 | 1 ng/ml, 10 ng/ml and 100 ng/ml | |
| Recombinant Human IL-17F | ImmunoTools | Cat.No.11349176 | 1 ng/ml, 10 ng/ml and 100 ng/ml | |
| Recombinant Human RANTES | ImmunoTools | Cat.No.11343196 | 0.3 ng/ml and 1.5 ng/ml | |
| Recombinant Human MIP-1α | ImmunoTools | Cat.No.11343206 | 1.5 ng/ml and 7.5 ng/ml | |
| Recombinant Human MIP-1β | ImmunoTools | Cat.No.11343223 | 1.0 ng/ml and 5 ng/ml | |
| Recombinant Human MCP | ImmunoTools | Cat.No.11343386 | 1 ng/ml, 10 ng/ml and 100 ng/ml | |
| Software, algorithm | FlowJo | BD Bioscences | version 10.6.1 | Software |
| BD FACSDiva Software | BD Biosciences | Software | ||
| Graphpad Prism | GraphPad Software LLC | v7.05 | Software |
Additional files
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Source data 1
Primary data for figures provided in manuscript.
- https://cdn.elifesciences.org/articles/52668/elife-52668-data1-v2.xlsx
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Supplementary file 1
PD-1 expressing cells for each subset expressed as percentage of live CD45+ cells, with range in parentheses.
- https://cdn.elifesciences.org/articles/52668/elife-52668-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52668/elife-52668-transrepform-v2.docx
