The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses

Abstract

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8⁺ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8⁺ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Keywords: B3GNT5; HLA class I; MHC class I; SPPL3; T cells; antigen presentation; glioma; glycosphingolipids; immune recognition; immunotherapy.

Figure 1.. A Haploid Genetic Screen Reveals SPPL3 as a Regulator of Antibody Accessibility to Membrane-Proximal HLA-I Regions

(A) Schematic overview of a genome-wide haploid genetic screen using HLA-I-specific W6/32 antibody. (B) Fish-tail plot showing per gene the mutation index (ratio of integrations mapped in the specified populations) against the amount of mapped integrations. A two-sided false discovery rate (FDR) (Benjamini-Hochberg) corrected Fisher’s exact test was applied. The symbol legend is indicated. (C) (Left) Titration curves of four HLA-I-specific antibodies on mixed barcoded HAP1 cells (see Figure S1E). The individual antibody binding epitopes are shown on the HLA-I structure. (Right) Flow cytometry histograms of nonsaturating antibody stain as indicated by the arrow (close to EC₅₀ values). MFI, mean fluorescence intensity. (D) Quantification of (C) using the ratio of WT and SPPL3^−/− EC₅₀ values. Mean ± SD is plotted (n = 5–8 independent experiments). (E) SPPL3-susceptibility of different epitopes is plotted on the HLA-I/β₂m crystal structure (individual epitopes in Figures 1C and S2A). See also Figures S1 and S2.

Figure 2.. SPPL3 Expression Promotes LIR-1 Binding to HLA-I and Enhances CD8⁺T Cell Activation

(A) CD8 and LIR-1 interaction sites mapped on the HLA-I/β₂m crystal structure. (B) IFN-γ production by T cells recognizing the specified endogenously derived antigens after coculture with the indicated HAP1 cells (n = 3). (C) Normalized ⁵¹Cr release (specific lysis) of the indicated HAP1 cells targeted by specified T cells at different effector-target (E:T) ratios (n = 3) (see also Figure S3A). (D) (Left) Representative titration curves of LIR-1 Fc fusion protein on indicated HAP1 cells (n = 2). (Right) Histogram of LIR-1 Fc binding at the indicated concentration (arrow). (E) Flow cytometry staining of HLA-C*05:01-transduced SPPL3^−/−HLA-A^−/−B^−/−C^−/− or depicted control HAP1 cells with nonsaturating concentrations of KIR2DL1 and KIR2DL2 Fc fusion proteins. Gray, unstained control. (F) Normalized quantification (median fluorescence intensity [median FI]) of HLA-I binding by the indicated fusion proteins (including data from D, E, and Figures 3G and 4E) (n = 3–6). (G) Predicted protein structure of SPPL3 with its catalytic residues magnified. (H) Histogram (cells from the RFP+ gate for transduced samples) and quantification (MFI normalized to the RFP− gate) of nonsaturating W6/32 stain on indicated HAP1 cells transduced with RFP-empty vector (EV), RFP-SPPL3, or catalytically inactive RFP-SPPL3 D271A (n = 5). See Figure S3C for B1.23.2 stain. (I) IFN-γ secretion in supernatant after coculture of the indicated T cells with RFP+ fluorescence-activated cell sorting (FACS)-sorted HAP1 SPPL3^−/− cells transduced as in (H) or with unsorted WT or HLA-I-deficient (gray) cells (n = 2–3). Mean ± SD of n independent experiments is plotted in (D), (F), and (H). A representative of n experiments is shown in (B), (C), and (I). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. See also Figure S3.

Figure 3.. SPPL3-Controlled GSLs Modulate Receptor Accessibility to HLA-I

(A–C) Schematic outlines of screening strategies to discover potential HLA-I regulators activated (A) or inactivated (C) by SPPL3. (B) Rocket plot of haploid genetic screens comparing the number of unique disruptive integrations per gene in BB7.2^lo- and W6/32^lo-sorted HAP1 populations. Two-sided FDR corrected Fisher’s exact test was applied. The symbol legend is indicated (see also Figure S4A). (D) Fish-tail plot of the haploid genetic screen in SPPL3^−/− HAP1 cells, showing per gene the mutation index (ratio of integrations mapped in indicated populations) against the amount of integrations. Statistics and color legend as in (B) (see also Figure S4B). (E) Schematic overview of the core enzymes involved in the synthesis of GSL subtypes. The putative SPPL3-targeted branch is shown in orange. PM, plasmamembrane. (F and G) Histograms of W6/32, B1.23.2 (F), and LIR-1 Fc (G) surface staining of the indicated HAP1 cells. Quantification (MFI, F; or median fluorescence intensity [median FI], G) is shown as mean ± SD (n = 3 independent experiments). Gray, unstained controls; S^−/−, SPPL3^−/−. ns, not significant; ** p<0.01; *** p<0.001. See also Figure S4.

Figure 4.. B3GNT5 Function Determines HLA-I Accessibility for Its Natural Receptors

(A) W6/32 surface staining of guide RNA (gRNA)-transduced (GFP+; gRNAs in Table S4) or control (GFP−) SPPL3^−/− HAP1 cells. (B and C) Quantification showing normalized MFIs of the depicted antibody binding to SPPL3^−/− (B) or WT (C) cells combined data from two experiments with 2–4 gRNAs per gene (examples in A) (Figures S4C and S4D). (D and E) Histograms and quantifications (MFI, D, or median fluorescence intensity, E, median FI) of surface staining of the indicated HAP1 cells using antibodies recognizing SPPL3-susceptible (W6/32, TP25.99, and ROU9A6) or SPPL3-independent (B1.23.2, WK4E3, and SN230G6) HLA-I epitopes (D; n = 4–7) or using LIR-1 Fc fusion protein (E; n = 2). S^−/−, SPPL3^−/−. (F) IFN-γ or GM-CSF secretion by depicted T cells after coculture with the indicated HAP1 cells. Representative of n = 3. Gray histograms are unstained controls. Mean ± SD of n independent experiments is plotted in (B)–(E). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. See also Figure S4.

Figure 5.. SPPL3 Controls the Generation of nsGSLs by Targeting B3GNT5

(A) Coimmunoprecipitation of the indicated FLAG-tagged proteins with RFP-EV (RFP), RFP-SPPL3 (R-SPPL3), or RFP-SPPL3 D271A (R-D271A). Sup, supernatant; TL, total lysate. Representative of n = 2. (B) Schematic model of B3GNT5 proteolysis by SPPL3. (C and D) TLC of the indicated HAP1 cell lysates incubated with UDP-GlcNAc and boron-dipyrromethene (BODIPY)-LacCer substrate to detect B3GNT5 activity (n = 3). BODIPY-Lc3Cer quantification (see Figure S5A for LC-MS validation) (C) and an example chromatogram (D) are shown. (E) Base peak chromatograms of porous graphitized carbon (PGC) LC-MS on total GSL glycans isolated from indicated HAP1 cells (n = 3). Proposed glycan structures and their relative abundance are listed in (F), Table S3 and Figure S5B. (F) Quantified relative abundance of the three major GSL types of the indicated cells. (G) Histograms and normalized MFI of cholera toxin B (CTB) and C3D-1 binding to indicated HAP1 cells (n = 3). Gray, unstained control; S^−/−, SPPL3^−/− (see Figure S5C). Mean ± SD of n independent experiments is plotted in (C), (F), and (G). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. See also Figure S5.

Figure 6.. Sialic Acid Residues on nsGSLs Are Required for HLA-I Shielding

(A) Quantified relative abundance of sialylated and fucosylated nsGSLs in WT or SPPL3^−/− HAP1 cells. Data from Figure 5E were reused. (B) Schematic view of targetable steps in nsGSL sialylation and fucosylation. NA, neuraminidase. (C) Antibody staining of the indicated HAP1 cells cultured with a serial dilution of sialyltransferase (SiaT) or fucosyltransferase (FucT) inhibitors. (D and E) Histograms (D) and normalized MFI (E) of the depicted antibody binding to HAP1 cells precultured with 100 μM inhibitors as in (C) (n = 6). (F) Histograms and quantification of the depicted antibody staining of the indicated HAP1 cells (n = 2). (G) Histograms and quantification of W6/32 and B1.23.2 binding to NA-treated HAP1 cells (n = 4). Gray histograms are unstained controls. S^−/−, SPPL3^−/−. Mean ± SD of n independent experiments is plotted in (A) and (E)–(G). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Figure 7.. Pharmacological Inhibition of GSL Synthesis in Glioma Enhances Anti-tumor Immune Responses

(A–C) TCGA-derived Kaplan-Meier curves showing the survival of patients with tumors expressing low or high B3GNT5 (A), low or high SPPL3 (B), or any of the four combinations thereof (C) (see Figure S7A). (D) Histograms of W6/32 and B1.23.2 binding to U-251 glioblastoma cells overexpressing GFP-SPPL3 or RFP-EV, combined in a single well. Quantification (MFIGFP+ cells/MFI RFP+ cells) includes a GFP-EV control (n = 5). (E) Histograms and normalized MFI of the depicted antibody staining of WT and UGCG^−/− U-251 cells (n = 4–5) (see Figure S7B). (F) IFN-γ secretion by the indicated T cells against WT or UGCG^−/− U-251 cells (n = 3). (G) Normalized MFI of the depicted antibody staining of the indicated HAP1 cells precultured with specified UGCG inhibitors (n = 2–7) (see Figures S7C–S7F). (H and I) IFN-γ or GM-CSF secretion by the indicated T cells cocultured with WT or SPPL3^−/− HAP1 cells that were precultured with or without the specified UGCG inhibitor (n = 3) (see Figure S7G for more T cell clones). (J) Histogram and normalized MFI of W6/32 binding to WT U-251 cells precultured with the indicated UGCG inhibitors (n = 3) (see Figure S7H).s (K) IFN-γ secretion by the indicated T cells after coculture with the depicted inhibitor-pretreated U-251 cells (n = 3). Gray histograms are unstained controls. Mean ± SD of n independent experiments is plotted in (D), (E), (G), and (J). A representative of n experiments is shown in (F), (H), (I), and (K). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. See also Figure S7.