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PD-1 agonism by anti-CD80 inhibits T cell activation and alleviates autoimmunity

Nature Immunology volume 23, pages 399–410 (2022)Cite this article

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Abstract

Targeted blockade of the checkpoint molecule programmed cell death 1 (PD-1) can activate tumor-specific T cells to destroy tumors, whereas targeted potentiation of PD-1 is expected to suppress autoreactive T cells and alleviate autoimmune diseases. However, the development of methods to potentiate PD-1 remains challenging. Here we succeeded in eliciting PD-1 function by targeting the cis-PD-L1–CD80 duplex, formed by binding of CD80 to the PD-1 ligand PD-L1, that attenuates PD-L1–PD-1 binding and abrogates PD-1 function. By generating anti-CD80 antibodies that detach CD80 from the cis-PD-L1–CD80 duplex and enable PD-L1 to engage PD-1 in the presence of CD80, we demonstrate that the targeted dissociation of cis-PD-L1–CD80 duplex elicits PD-1 function in the condition where PD-1 function is otherwise restricted. We demonstrate using murine models that the removal of PD-1 restriction is effective in alleviating autoimmune disease symptoms. Our findings establish a method to potentiate PD-1 function and propose the removal of restraining mechanisms as an efficient strategy to potentiate the function of inhibitory molecules.

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Fig. 1: Anti-CD80 TKMG48 enables PD-1 to associate with PD-L1 in the presence of CD80 by liberating PD-L1 from cis-PD-L1–CD80 duplex.
Fig. 2: Anti-CD80 TKMG48 enables PD-1 to inhibit T cell activation in the presence of CD80.
Fig. 3: Anti-human CD80 TKMF5 enables human PD-1 to inhibit T cell activation in the presence of human CD80 by dissociating cis duplex of human PD-L1 and CD80.
Fig. 4: Anti-CD80 TKMG48 augments PD-1-binding capacities of DCs and suppresses antigen-specific T cell responses in vivo.
Fig. 5: Alleviation of pGPI-induced experimental arthritis by TKMG48.
Fig. 6: Alleviation of arthritis in SKG mice by TKMG48.
Fig. 7: Alleviation of experimental autoimmune encephalomyelitis by TKMG48.
Fig. 8: Alleviation of Sjögren’s syndrome-like diseases in NFS/sld mice by TKMG48.

Data availability

All data in the present study are available within the article. Source data are provided with this paper.

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Acknowledgements

We thank T. Kurosaki (IIA1.6), T. Kitamura (Plat-E), L. Ignatowicz (BW-1100.129.237), K. Murphy (DO11.10 TCR transgenic mouse) and T. Honjo (DO11.10 T, 2.4G2, J43, Sp2/O and 293T) for kindly providing materials; H. Tsuduki, Y. Okamoto, M. Aoki, J. Tsueda, A. Mikuniya, R. Matsumura and M. Saito for technical and secretarial assistance; and the other members of our laboratory for helpful discussions. This work was supported in part by grants from the Basic Science and Platform Technology Program for Innovative Biological Medicine of the Japan Agency for Medical Research and Development (JP18am0301007 to T.O.), Grant-in-Aid by the Japan Society for the Promotion of Science (JP18H05417 to T.O.; JP19H01029 to T.O.; JP21K06526 to D.S.), the Naito Foundation (to T.O.) and Joint Usage and Joint Research Programs by the Institute of Advanced Medical Sciences of Tokushima University (to T.O. and T.T.).

Author information

Authors and Affiliations

  1. Laboratory of Molecular Immunology, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan

    Daisuke Sugiura, Il-mi Okazaki, Takumi Maruhashi, Kenji Shimizu & Taku Okazaki

  2. Laboratory for Immune Regulation, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan

    Daisuke Sugiura, Il-mi Okazaki, Takeo K. Maeda, Takumi Maruhashi, Kenji Shimizu & Taku Okazaki

  3. Department of Oral Molecular Pathology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan

    Rieko Arakaki & Naozumi Ishimaru

  4. Laboratory for Embryology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan

    Tatsuya Takemoto

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  1. Daisuke Sugiura
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Contributions

D.S. and T.O. designed the experiments. D.S., I.O., T.K.M., T.M. and K.S. established experimental systems and performed the in vivo and in vitro experiments using cultured cells and mice. D.S. and T.T. generated gene-targeted mice. R.A. and N.I. performed histological analyses. D.S. and T.O. wrote the manuscript with all authors contributing to writing. T.O. supervised the entire project.

Corresponding author

Correspondence to Taku Okazaki.

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Competing interests

D.S., T.K.M. and T.O. have a pending patent of an immunosuppressive method related to the immunoregulatory mechanism reported here (WO 2020/116636). T.O. received a research grant from Ono Pharmaceutical. The other authors declare no competing interests.

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Nature Immunology thanks Dario Vignali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement: Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended data Fig. 1 Effects of PD-L1- and PD-L2-deficiences on the TKMG48-induced binding of PD-1-EC to DCs.

a,b, Generation of C57BL/6N-Pdcd1lg2–/– mice. Nucleotide and deduced amino acid sequences of PD-L2 gene in C57BL/6N-Pdcd1lg2–/– mice (a). Sequences of guide RNA (gRNA) and protospacer adjacent motif (PAM) are indicated with black and green lines, respectively. In C57BL/6N-Pdcd1lg2–/– mice, a premature stop codon is expected to be introduced by the insertion of 1 nucleotide that is colored in red. Lack of PD-L2 expression on LPS-activated splenic CD8α+ and CD11b+ DCs from Pdcd1lg2–/– mice (b). c, PD-1 binding capacities of DCs from mice deficient for PD-L1 (Cd274–/–) and PD-L2 (Pdcd1lg2–/–) in the presence of the indicated amount of TKMG48 or 30 µg ml−1 of Ctrl Ig. Representative histogram plots of 3 (b) and 2 (c) independent experiments are shown.

Extended data Fig. 2 Characterization of TKMG48-binding region of CD80.

a, Recognition of the IgV-like domain of CD80 by TKMG48. Bindings of TKMG48 and 16-10A1 (another clone of anti-CD80 Ab) to DOdPL cells expressing indicated chimeric molecules of CD80 and CD86 are shown. b, Attenuation of TKMG48 binding to CD80 by the introduction of amino acid substitutions that abrogate cis-PD-L1-CD80 interactions. Bindings of TKMG48 and 16-10A1 to DOdPL cells expressing indicated CD80 mutants are shown. c, No competition between TKMG48 and 16-10A1 on CD80 binding. The bindings of TKMG48 and 16-10A1 to DOdPL cells pre-incubated with the indicated Abs (10 µg ml−1) are shown. Representative data of 3 independent experiments are shown.

Extended data Fig. 3 Effects of TKMG48 on PD-1 and CTLA-4 signals.

a,b, Requirement of PD-1-signal for the TKMG48-induced inhibition of T cell activation. Expression of PD-1 and its mutants on DOdP T cells (a). DOdP T cells expressing PD-1 or its mutants were stimulated with pOVA323-339 (0.3 μM) using IIAdL cells expressing indicated molecules as APCs in the presence or absence of TKMG48 (10 μg ml−1). c, No substantial effect of TKMG48 on CTLA-4 signal. DOdP or DOdP-CTLA-4 T cells were stimulated with IIAdL-CD80 or IIAdL-CD80:CD86 cells pulsed with pOVA323-339 (0.3 µM) in the presence of TKMG48 (10 µg ml−1). IL-2 concentrations of the culture supernatants are shown (b,c). Representative data of 2 (a,b) and 3 (c) independent experiments are shown.

Source data

Extended data Fig. 4 Dose-dependent effects of PD-L1, CD80, and PD-1 on TKMG48-induced T cell suppression.

a–d, Dose-dependent effects of PD-L1 and CD80 on TKMG48-induced T cell suppression. Expression of PD-L1 and CD80 at high, intermediate (int), and low levels on IIAdL cells (a). PD-1-EC-binding intensities of IIAdL cells expressing PD-L1 and CD80 at varying levels in the presence or absence of TKMG48 (10 μg ml−1) (b). TKMG48-induced PD-1-mediated inhibition of T cell activation in a manner dependent on the amount of PD-L1 on IIAdL cells (c,d). DOdP-PD-1 T cells were stimulated with pOVA323-339 (0.3 μM) using IIAdL cells expressing indicated molecules at indicated levels as APCs in the presence or absence of anti-PD-L1 Ab (2 μg ml−1) and TKMG48 (10 μg ml−1). e–g, Effects of PD-1 expression level on TKMG48-induced T cell suppression. Expression of PD-1 at high, int, and low levels on DOdP T cells (e). Inhibition of T cell activation in a manner dependent on the amount of PD-1 on T cells (f). PD-1-mediated inhibition of T cell activation by TKMG48 in a manner dependent on the amount of PD-1 on T cells (g). DOdP T cells expressing PD-1 at indicated level were stimulated with pOVA323-339 (0.3 μM) using IIAdL cells expressing indicated molecules as APCs (f,g). TKMG48 or Ctrl Ig (10 µg ml−1) was added as indicated (g). IL-2 concentrations of the culture supernatants (c,f,g) and the percentages of inhibition (d) are shown. Representative data of 3 independent experiments are shown. Error bars denote SEM.

Source data

Extended data Fig. 5 TKMG48-induced PD-1-mediated inhibition of cytokine production and proliferation of primary T cells.

a,b, TKMG48-induced PD-1-mediated inhibition of cytokine production from primary T cells upon antigen stimulation. Primary T cells from DO11.10 transgenic mice were stimulated with the indicated amount of pOVA323-339 using IIAdL cells expressing indicated molecules as APCs in the presence or absence of anti-PD-L1 Ab (3 µg ml−1) and TKMG48 (10 µg ml−1) for 72 hours. Concentrations of IL-2 (a) and IFN-γ (b) are shown. c, TKMG48-induced PD-1-mediated inhibition of proliferation of primary T cells upon antigen stimulation. Primary T cells from DO11.10 transgenic mice were labeled with CFSE and stimulated with pOVA323-339 (0.3 nM) using IIAdL-CD80:CD86:PD-L1 cells as APCs in the presence or absence of anti- PD-L1 Ab (3 µg ml−1) and TKMG48 (10 µg ml−1) for 72 hours and analyzed by flowcytometry for the dilution of CFSE. Representative histogram plots and the frequencies of cells with more than 2 and 3 cell divisions are shown. Representative data of 3 independent experiments are shown. Unpaired two-tailed Student’s t-test (d). **p < 0.01. Error bars denote SEM.

Source data

Extended data Fig. 6 Characterization of TKMF5-binding region of hCD80.

a, Defective binding of TKMF5 to hCD80 mutants that cannot form cis-PD-L1-CD80 duplex. Bindings of TKMF5 and 2D10 (another clone of anti-hCD80 Ab) to DOdPL cells expressing hCD80 with or without I92E/L104E mutation are shown in histogram plots. b,c, No substantial effect of TKMF5 on hCD80–hCTLA-4 and hCD80–hCD28 bindings. Binding capacities of hCTLA-4-EC and hCD28-EC to IIAdL-hCD80 cells were evaluated in the presence of the indicated amount of TKMF5 and 2D10. Representative histogram plots (b) and the percentages of hCTLA-4-EC- and hCD28-EC-positive cells (c) are shown. Representative data of 3 independent experiments are shown.

Source data

Extended data Fig. 7 TKMF5-induced PD-1-mediated inhibition of cytokine production from Jurkat human T cell lymphoma cells.

a, Expression of hPD-1 on Jurkat-HA1.7 cells with or without its overexpression. b, Expression of hPD-L1, hCD80, and hCD86 and the PD-1-EC-binding intensities of Raji-DR1 cells reconstituted with indicated molecules. c, Restriction of hPD-1 function by cis-hPD-L1-hCD80 interactions in the activation of Jurkat-HA1.7 cells. IL-2 concentrations of the culture supernatants are shown. d,e, Dose-dependent effects of TKMF5 on hPD-1-EC bindings to hPD-L1 in the presence of hCD80. Binding capacities of hPD-1-EC to Raji-DR1-hPD-L1:hCD80 cells were evaluated in the presence of the indicated amount of TKMF5 or 100 µg ml−1 of Ctrl Ig. Representative histogram plots (d) and the percentages of hPD-1-EC-positive cells (e) are shown. f–i, Elicitation of hPD-1 function by TKMF5. Jurkat-HA1.7 cells with or without hPD-1 were stimulated with pHA307-319-pulsed (1 µM) Raji-DR1 cells expressing hCD80 and hPD-L1 at the indicated combinations in the absence (f,g) or presence (h,i) of hCD86. Indicated amounts of TKMF5 or 30 µg ml−1 of Ctrl Ig were added. IL-2 concentrations of the culture supernatants are shown (f,h). The percentages of inhibition in 3 independent experiments are summarized (g,i). Unpaired two-tailed Student’s t-test (g,i). **p < 0.01; ***p < 0.001. Error bars denote SEM. Representative data of 3 independent experiments are shown.

Source data

Extended data Fig. 8 Abrogation of the therapeutic effects of TKMG48 on pGPI-induced experimental arthritis by the blockade of PD-L1-PD-1 binding.

a,b, Therapeutic effects of TKMG48 on pGPI-induced arthritis starting after the full manifestation of the disease. Experimental design (a). Clinical scores of arthritis in mice (n = 10 each, female, 7-week-old) treated with or without TKMG48 (b). c–g, Abrogation of the therapeutic effects of TKMG48 by the blockade of PD-L1-PD-1 binding. Experimental design (c). Female 6-week-old mice were used (n = 8 each). Comparison of therapeutic effects of TKMG48 on arthritic symptoms of mice treated with blocking Abs against PD-L1 (d,f) or PD-1 (e,g). Exacerbations of arthritis by blocking Abs against PD-L1 (f) or PD-1 (g) were evaluated. Two-way repeated measure ANOVA with Tukey’s HSD post hoc test (b,d–g). ***p < 0.001. Error bars denote SEM.

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Extended data Fig. 9 Suppression of CD4+ T cells with IFN-γ-, IL-17A-, and GM-CSF-producing capacities by TKMG48 in SKG mice.

T cells in popliteal LNs of SKG mice were analyzed 70 days after the treatment of Zymosan for their expression of CD44 and CD62L as well as the capacities to produce IFN-γ, IL-17A, and GM-CSF upon stimulation with PMA (50 ng ml−1) and ionomycin (500 ng ml−1) for 4 hours in the presence of brefeldin A. Representative contour plots are shown for the expression of CD44 and CD62L on CD4+ T cells from SKG mice treated with or without TKMG48 (n = 10 each, female, 7-week-old) (a). Total numbers of cells and frequencies and numbers of CD44high CD62Llow CD4+ T cells (b) and numbers of CD4+ T cells with IFN-γ, IL-17A, and GM-CSF producing capacities (c) in popliteal LNs of SKG mice treated with or without TKMG48 are shown. Unpaired two-tailed Student’s t-test (b,c). **p < 0.01; ***p < 0.001.

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Extended data Fig. 10 Gating strategies for DCs.

a, Mouse splenic DCs. Splenocytes stimulated with LPS were stained with indicated Abs to distinguish CD8α+ and CD11b+ DCs. b, DCs infiltrating in spinal cords of EAE-induced mice. Leukocytes isolated from spinal cords of EAE-induced mice were stained with indicated Abs to distinguish CD11b+ DCs.

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Sugiura, D., Okazaki, Im., Maeda, T.K. et al. PD-1 agonism by anti-CD80 inhibits T cell activation and alleviates autoimmunity. Nat Immunol 23, 399–410 (2022). https://doi.org/10.1038/s41590-021-01125-7

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