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LAG-3 inhibits the activation of CD4+ T cells that recognize stable pMHCII through its conformation-dependent recognition of pMHCII

Nature Immunology volume 19, pages 1415–1426 (2018)Cite this article

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Abstract

The success of tumor immunotherapy targeting the inhibitory co-receptors PD-1 and CTLA-4 has indicated that many other co-receptors might be potential druggable targets, despite limited information about their functional differences. Here we identified a unique target selectivity for the inhibitory co-receptor LAG-3 that was intrinsic to its immunoregulatory roles. Although LAG-3 has been reported to recognize major histocompatibility complex (MHC) class II, it did not recognize MHC class II universally; instead, we found that it selectively recognized stable complexes of peptide and MHC class II (pMHCII). LAG-3 did not directly interfere with interactions between the co-receptor CD4 and MHC class II or between the T cell antigen receptor and MHC class II. Instead, LAG-3 preferentially suppressed T cells responsive to stable pMHCII by transducing inhibitory signals via its intracellular region. Thus, LAG-3 might function more selectively than previously thought and thereby maintain tolerance to dominant autoantigens.

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Fig. 1: Identification of MHC class II–expressing cells with lower LAG-3-binding capacity.
Fig. 2: Requirement for LAG-3-binding capacity in APCs for the elicitation of its inhibitory function.
Fig. 3: Selective binding of LAG-3 to CIITA-induced MHC class II.
Fig. 4: Conformation-dependent binding of LAG-3 to stable pMHCII.
Fig. 5: Inhibitory effect of LAG-3 at the peptide-specific level.
Fig. 6: Suppression of diabetogenic CD4+ T cells recognizing stable autoantigen–MHC class II by LAG-3 in NOD mice.
Fig. 7: Weak inhibitory effect of LAG-3 at APC type–specific level.
Fig. 8: Requirement for the intracellular region for the inhibitory effect of LAG-3.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank H. Kozono and O. Kanagawa for critical reading of the manuscript; L. Ignatowicz, M. Hattori, T. Kurosaki, and T. Kitamura for kindly providing BW-1100.129.237, CH27, IIA1.6, and Plat-E cells, respectively; T. Honjo for kindly providing DO11.10, 2B4.11, WEHI-231, P815, and SP2/o cells; K. Murphy for kindly providing DO11.10 Tg mice; A. Otsuka, Y. Okamoto, M. Aoki, and H. Tsuduki for technical assistance; and the other members of our laboratory for helpful discussions. This work was supported in part by the Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Agency, Basic Science and Platform Technology Program for Innovative Biological Medicine of the Japan Agency for Medical Research and Development (JP18am0301007), Grant-in-Aid by the Japan Society for the Promotion of Science (JP18H05417), the Tokyo Biochemical Research Foundation, and Uehara Memorial Foundation.

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  1. Division of Immune Regulation, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan

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

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Contributions

T.M., I.O., and T.O. designed and analyzed the experiments. T.M., I.O., S.T., D.S., T.K.M., and K.S. performed experiments. T.M., I.O., D.S., T.K.M., and K.S. performed the staining and functional experiments using cultured cells. T.M. and S.T. generated gene-targeted cells. T.M. performed the staining experiments of primary cells and functional experiments using NOD mice. I.O. performed the expression cloning. T.M. and T.O. wrote the manuscript with all authors contributing to writing. T.O. oversaw the entire project.

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Correspondence to Taku Okazaki.

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Supplementary Figure 1 Mouse LAG-3-P111A mutant lacking inhibitory and binding capacities.

a, Amino acid sequence alignment of the mouse and human LAG-3 D1 domain. Residues substituted with alanine and proline 111 are indicated in blue and red, respectively. b,c, Inhibitory function of mouse LAG-3 mutants. DO11.10 T cells expressing EGFP (Mock), mLAG-3-WT, or mLAG-3 mutants with alanine substitution were stimulated with IIA1.6 cells pulsed with OVA323–339 peptide in the presence or absence of anti-LAG-3 blocking Ab (TKB58) and the concentration of IL-2 in the culture supernatant was measured by ELISA (b). Percent inhibition of IL-2 production by mouse LAG-3-WT and -mutants is shown (c). d, Comparable surface expression levels of LAG-3 and LAG-3-P111A. EGFP (Mock), LAG-3, and LAG-3-P111A were retrovirally transduced into DO11.10 T cells and the surface expression levels were determined by flow cytometric analysis using anti-LAG-3 Abs specific to D1 domain (TKB58) and D2 domain (C9B7W). e, Disappearance of the binding capacity of LAG-3-EC to IIA1.6 cells by P111A substitution. Binding intensities of pentameric LAG-3-EC and LAG-3-P111A-EC to IIA1.6 cells were determined by flow cytometric analysis. Bar graphs indicate the mean of technical duplicates in one representative experiment (b,c). Gray shaded histogram represents the negative control stained with the secondary Ab (e). Data are representative of two (b,c), or more than three independent experiments (d,e).

Supplementary Figure 2 Gating strategy for mouse primary B cells and DCs in flow cytometric analysis.

Bone marrow cells and splenocytes were stained with the indicated Abs to be divided into indicated subpopulations. MHCII was detected by M5/114.15.2 that does not affect the LAG-3-EC binding to cells at the working concentration for flow cytometry (0.07 μg/ml). MZ, marginal zone B cell; MZP, marginal zone precursor B cell; FOL I, follicular type I B cell; FOL II, follicular type II B cell; pDC, plasmacytoid dendritic cell; cDC, conventional dendritic cell. Data are representative of three independent experiments.

Supplementary Figure 3 Conferment of the capacity to exert LAG-3 inhibitory function on BW5147 cells by CIITA.

a, LAG-3-EC binding intensities and MHCII expression levels of BW5147-I-Ad cells with or without retroviral transduction of CIITA. Overexpression of CIITA conferred the LAG-3 binding capacity to BW5147-I-Ad cells as well as induced the expression of endogenous I-Ak and I-Ek. b,c, Exertion of LAG-3 inhibitory function by BW5147-I-Ad cells overexpressing CIITA. DO11.10 T cells expressing EGFP (Mock) or LAG-3 were stimulated with indicated cells pulsed with indicated amount of OVA323−339 peptide. IL-2 concentration (b) and LAG-3-mediated inhibitory effect (c) are shown. d, Requirement of I-Ak or I-Ek for the LAG-3-EC binding to BW5147-CIITA cells. LAG-3-EC binding intensities and MHCII expression levels of BW5147-CIITA cells lacking I-Ak and I-Ek generated by using CRISPR/Cas9 system. Each symbol represents an individual technical replicate (c, n = 4). Bar graphs indicate the mean of technical duplicates in one representative experiment (b) or the mean ± s.d. of technical quadruplicates pooled from two independent experiments (c). ND, not detected. Two-tailed paired Student’s t-test (d,f). ***P < 0.005. Data are representative of more than three independent experiments (a,b,d).

Supplementary Figure 4 Induction of Ii and H2-DM by CIITA, expression of Ii and H2-DM on LAG-3-binding cells, and lack of Ii and H2-DM expression in cells with genetic deletion of the corresponding gene.

a, Gene expression of MHCII accessory molecules in P815-CIITA and IIA1.6 cells relative to parental P815 cells analyzed by qPCR. Values were normalized to the expression of Actb. b, Reduced and completely abolished expression of Ii and H2-DM, respectively in IIA1.6-CIITA KO cells. Intracellular expression of Ii and H2-DM in IIA1.6-CIITA KO cells was determined by flow cytometric analysis. c, Lower expression of Ii and H2-DM in LAG-3-EC-low/negative cell populations compared with LAG-3-EC-high populations shown in Fig. 1b. Relative expression levels (geoMFI) of Ii, H2-DM and MHCII in bone marrow (BM) mature B cells and spleen MZP B cells (left), and LAG-3-EC-low/negative and -high populations in spleen CD8+ cDCs (right) were determined by flow cytometric analysis. d, The defective expression of Ii and H2-DM on IIA1.6 cells in which genes encoding Ii and H2-DMα, respectively were knocked-out by using CRISPR/Cas9 system. Each symbol represents an individual mouse (c). Bar graphs indicate the mean of technical triplicates in one representative experiment (a) or the mean ± s.e.m. of 3 mice in one representative experiment (c). Two-tailed Mann-Whitney U-test (c). *P < 0.05, ***P < 0.005. Data are representative of two (a,c) or three (b,d) independent experiments.

Supplementary Figure 5 Accumulation of LAG-3 at the interface between T cells and APCs expressing stable but not unstable pMHCII.

a–e, Confocal microscopy of LAG-3-EGFP and LAG-3-P111A-EGFP on DO11.10 T cells co-cultured with indicated cell lines expressing endogenous or exogenous I-Ad (a), expressing CIITA (b), expressing exogenous I-Ad and non-cognate stable or unstable pMHCII (c–e) pulsed with OVA323−339 peptide. Scale bars, 10 μm. Images are representative of more than two independent experiments (a–e). White arrowheads indicate the accumulation of LAG-3-EGFP at the interface of T cells and APCs.

Supplementary Figure 6 Dose-dependent binding and low dissociation rate of pentameric human LAG-3-EC and human CD4-EC.

a,b, LAG-3-EC binding intensities and MHCII expression levels of Jurkat cells with or without retroviral transduction of human CIITA (a) and BW5147 cells expressing HLA-DR1 covalently linked with pHA307–318 (b). Overexpression of human CIITA induced the expression of endogenous HLA-DR and HLR-DQ in Jurkat cells and conferred the LAG-3 binding capacity. Expression of HLA-DR1 covalently linked with dominant epitope of influenza HA (pHA307–318) conferred the LAG-3 binding capacity to BW5147 cells. c,d, Dose-dependent binding of hLAG-3-EC and hCD4-EC to Raji and Jurkat-hCIITA cells. Raji and Jurkat-hCIITA cells were incubated with hLAG-3-EC (WT) and hLAG-3-P115A-EC (P115A) (c) or hCD4-EC (d) at indicated concentrations, and their bindng was detected by streptavidin or anti-DYKDDDDK Tag Ab, respectively. The biniding intensities (geoMFI) of solublue proteines were determined by flow cytometric analysis. The dose-dependent binding curves were obtained by fitting the data to the Hill equation. The bindings of hLAG-3-EC and hCD4-EC to Raji and Jurkat-hCIITA cells were saturated at 20 and 5 μg/ml, respectively. e, Slow disscociation rate of hLAG-3-EC and hCD4-EC from Jurkat-hCIITA cells. Jurkat-hCIITA cells were incubated with hLAG-3-EC or hCD4-EC at saturating concentrations, follwed by the staining with streptavidin or anti-DYKDDDDK Ab, respectively. The biniding intensities (geoMFI) of solublue proteines after the subsequent incubation for the indicated time period were determined by flow cytometric analysis. The percentage of binding (Log10 geoMFI) was calculated relative to the binding at 0 min. Data are representative of two independent experiments (a–e).

Supplementary Figure 7 CD4 dependency of DO11.10, 2B4.11, and BWCD4-3A9 TCR cells for activation.

a, Surface expression levels of endogenous or exogenous CD4 on the indicated cell lines. b, CD4-dependent IL-2 production of BWCD4-3A9 TCR cells. Indicated cell lines were stimulated with indicated cells pulsed with the indicated amount of antigenic peptides in the presence of rat IgG2b isotype control (RTK4530) or anti-CD4 blocking Ab (GK1.5), and the concentration of IL-2 in the culture supernatant was measured by ELISA. Bar graphs indicate the mean of technical duplicates in one representative experiment. c, Comparable surface expression levels of LAG-3 and its mutant lacking the intracellular region (ΔIC). LAG-3 and LAG-3-ΔIC were overexpressed in indicated cell lines under the control of chicken beta-actin promoter and LTR retroviral promoter, respectively. ND, not detected. Data are representative of more than two independent experiments (a–c).

Supplementary Figure 8 Schematic representations of the LAG-3-mediated inhibition of T cell activation at peptide- and APC-type-specific levels and the inhibitory mechanism of LAG-3.

a, LAG-3 strongly inhibits the activation of T cells recognizing stable pMHCII by binding to stable pMHCII (left). LAG-3 can also partially inhibit the activation of T cells recognizing unstable pMHCII and pMHCI, when APCs express stable non-cognate pMHCII abundantly (right). b, Current findings indicate that LAG-3 inhibits T cell activation by transducing inhibitory signal via its intracellular region (left) but not by attenuating the CD4-MHCII (middle) or the TCR-pMHCII (right) interactions.

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Maruhashi, T., Okazaki, Im., Sugiura, D. et al. LAG-3 inhibits the activation of CD4+ T cells that recognize stable pMHCII through its conformation-dependent recognition of pMHCII. Nat Immunol 19, 1415–1426 (2018). https://doi.org/10.1038/s41590-018-0217-9

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