Abstract
Background CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. Methods To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. Results Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A′-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. Conclusion Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.
Original language | English |
---|---|
Article number | e007631 |
Journal | Journal for Immunotherapy of Cancer |
Volume | 11 |
Issue number | 12 |
DOIs | |
Publication status | Published - 1 Dec 2023 |
Keywords
- Annexin A5
- Hepatitis A Virus Cellular Receptor 2/metabolism
- Humans
- Phosphatidylserines/metabolism
- Single-Domain Antibodies/metabolism
- T-Lymphocyte Subsets
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In: Journal for Immunotherapy of Cancer, Vol. 11, No. 12, e007631, 01.12.2023.
Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
T1 - Enhanced CD1d phosphatidylserine presentation using a single-domain antibody promotes immunomodulatory CD1d-TIM-3 interactions
AU - Lameris, Roeland
AU - Shahine, Adam
AU - Veth, Myrthe
AU - Westerman, Bart
AU - Godfrey, Dale I.
AU - Hulsik, David Lutje
AU - Brouwer, Patricia
AU - Rossjohn, Jamie
AU - de Gruijl, Tanja D.
AU - van der Vliet, Hans J.
N1 - Funding Information: 1Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands 2Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia 3Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands 4Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia 5LAVA Therapeutics, Utrecht, The Netherlands 6Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK Contributors Conceptualization: RL, AS, DIG, JR, TDdG, HJvdV. Methodology: RL, AS, BW, DLH. Investigation: RL, AS, MV, BW, DLH, PB. Visualization: RL, AS, HJvdV. Resources: BW. Funding acquisition: DIG, JR, TDdG, HJvdV. Supervision: DIG, JR, TDdG, HJvdV. Writing original draft: RL, AS, TDdG, HJvdV. Guarantor: HJvdV Funding This work was supported by CCA-VICI grant no. 2000483 from the VU University Medical Center, grant no. 140343 from Worldwide Cancer Research, funding from LAVA Therapeutics (RL, MV, DLH, PB and HJvdV), the National Health and Medical Research Council of Australia (NHMRC; grant nos. 1113293 and 1140126), and the Cancer Council of Victoria (JR and DIG). DIG was supported by an NHMRC Senior Principal Research Fellowship (no. 1117766) and subsequently by an NHMRC Investigator Award (2008913). JR is supported by an NHMRC Investigator award. AS is supported by an ARC DECRA Fellowship (DE210101031). We thank the Monash Macromolecular Crystallization Facility staff for assistance with crystallization, the Australian Synchrotron for assistance with data collection, the University of Melbourne, Department of Microbiology and Immunology Flow Cytometry facility for flow cytometry support, and Dr Adam P Uldrich for providing the SKW-3.NKT12 and SKW-3.NKT15 cells and the C1R.GFP cells expressing WT CD1d or mutant CD1d. Funding Information: This work was supported by CCA-VICI grant no. 2000483 from the VU University Medical Center, grant no. 140343 from Worldwide Cancer Research, funding from LAVA Therapeutics (RL, MV, DLH, PB and HJvdV), the National Health and Medical Research Council of Australia (NHMRC; grant nos. 1113293 and 1140126), and the Cancer Council of Victoria (JR and DIG). DIG was supported by an NHMRC Senior Principal Research Fellowship (no. 1117766) and subsequently by an NHMRC Investigator Award (2008913). JR is supported by an NHMRC Investigator award. AS is supported by an ARC DECRA Fellowship (DE210101031). We thank the Monash Macromolecular Crystallization Facility staff for assistance with crystallization, the Australian Synchrotron for assistance with data collection, the University of Melbourne, Department of Microbiology and Immunology Flow Cytometry facility for flow cytometry support, and Dr Adam P Uldrich for providing the SKW-3.NKT12 and SKW-3.NKT15 cells and the C1R.GFP cells expressing WT CD1d or mutant CD1d. Funding Information: Competing interests DLH, PB and HJvdV are employees of LAVA Therapeutics, a company that develops bispecific gamma-delta T-cell engagers, and own LAVA Therapeutics shares and/or stock options. RL, TDdG and HJvdV are named inventors on international patent application WO 2016/122320 Al (‘Single domain antibodies targeting CD1d’) which partially relates to the work described in this paper. TDdG is a consultant for and a shareholder of LAVA Therapeutics. RL and MV were funded by a LAVA Therapeutics grant to Amsterdam UMC. JR has received funding from LAVA Therapeutics. DIG is a member of the scientific advisory board and a shareholder of Avalia Immunotherapies, a company working on NKT cell-based vaccines and is an inventor on patent applications WO 2021/127745 and WO 2021/127744 that describe effects of cross-linking CD1 molecules and WO2020/231274 that describes CD1d-dependent glycopeptide vaccines. Publisher Copyright: © Author(s) (or their employer(s)) 2023.
PY - 2023/12/1
Y1 - 2023/12/1
N2 - Background CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. Methods To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. Results Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A′-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. Conclusion Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.
AB - Background CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. Methods To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. Results Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A′-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. Conclusion Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.
KW - Annexin A5
KW - Hepatitis A Virus Cellular Receptor 2/metabolism
KW - Humans
KW - Phosphatidylserines/metabolism
KW - Single-Domain Antibodies/metabolism
KW - T-Lymphocyte Subsets
UR - http://www.scopus.com/inward/record.url?scp=85178345397&partnerID=8YFLogxK
U2 - https://doi.org/10.1136/jitc-2023-007631
DO - https://doi.org/10.1136/jitc-2023-007631
M3 - Article
C2 - 38040419
SN - 2051-1426
VL - 11
JO - Journal for Immunotherapy of Cancer
JF - Journal for Immunotherapy of Cancer
IS - 12
M1 - e007631
ER -