Tumor-Expressed SPPL3 Supports Innate Antitumor Immune Responses

Abstract

The development of an effective antitumor response relies on the synergistic actions of various immune cells that recognize tumor cells via distinct receptors. Tumors, however, often manipulate receptor-ligand interactions to evade recognition by the immune system. Recently, we highlighted the role of neolacto-series glycosphingolipids (nsGSLs), produced by the enzyme β1,3-N-acetylglucosaminyltransferase 5 (B3GNT5), in tumor immune escape. We previously demonstrated that loss of signal peptide peptidase like 3 (SPPL3), an inhibitor of B3GNT5, results in elevated levels of nsGSLs and impairs CD8 T cell activation. The impact of loss of SPPL3 and an elevated nsGSL profile in tumor cells on innate immune recognition remains to be elucidated. This study investigates the antitumor efficacy of neutrophils, NK cells, and γδ T cells on tumor cells lacking SPPL3. Our findings demonstrate that SPPL3-deficient target cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and γδ T cells. Mechanistically, SPPL3 influences trogocytosis and γδ T cell-instigated killing through modulation of nsGSL expression, whereas SPPL3-mediated reduced killing by NK cells is nsGSL-independent. The nsGSL-dependent SPPL3 sensitivity depends on the proximity of surface receptor domains to the cell membrane and the affinity of receptor-ligand interactions as shown with various sets of defined antibodies. Thus, SPPL3 expression by tumor cells alters crosstalk with immune cells through the receptor-ligand interactome thereby driving escape not only from adaptive but also from innate immunity. These data underline the importance of investigating a potential synergism of GSL synthesis inhibitors with current immune cell-activating immunotherapies.

FIGURE 1

nsGSLs impair antitumor responses of γδ T cells. Freshly expanded γδ T cells from four donors were incubated with HAP1 cells (WT, SPPL3^−/−, and SPPL3^−/−B3GNT5^−/−) for 5 h in a 1:1, 5:1, and 10:1 effector‐to‐target (E:T) ratio. The target cells were either untreated or pretreated with PAM O/N prior to culture. Triplicates were used for each donor. Representative flow cytometry plots (A) and combined data of n = 4 (B) showing the percentage of dead target cells (near‐IR positive) without and with addition of γδ T cells in different ratios. Data shown are representative from four experiments with four donors per experiment and three technical replicates per datapoint. A one‐way ANOVA was used to assess statistical significances. PAM, pamidronate.

FIGURE 2

SPPL3 regulates NK cell immune responses independent of nsGSLs. NK cells were cocultured with NALM6 or K562 target cells for 5 h in different E:T ratios. (A) Representative flow cytometry plots and (B) combined data illustrating the proportion of dead K562 WT and SPPL3^−/− cells (near‐IR positive) cocultured with NK cells in a 0:1, 1:1, and 5:1 ratio. Data shown are representative from three experiments with four donors per experiment and three technical replicates per datapoint. Representative flow cytometry plots (C) and combined data of n = 2 (D) showing the percentage of dead (near‐IR positive) NALM6 WT or SPPL3^−/− cells with different E:T ratios. Data shown are representative from three experiments with two donors per experiment and three technical replicates per datapoint. (E and F) Extracted ion chromatograms of total GSL glycan content released from (E) NALM6 WT and SPPL3^−/− cells as well as (F) K562 WT, UGCG^−/−, and SPPL3^−/− cells using PGC LC–MS. A paired Student's T‐test (B) or a one‐way ANOVA (D) was used to assess statistical significances.

FIGURE 3

Trogocytosis of HAP1 SPPL3^−/− cells is diminished due to nsGSLs. (A) Histogram illustrating ICAM1 staining of HAP1 WT, SPPL3^−/−, and SPPL3^−/−B3GNT5^−/− cells with a saturating antibody dilution (1:10, left plot) and non‐saturating dilution (1:100, right plot). The data represent two independent experiments with three technical replicates. Trogocytosis of HAP1 cells (stained with a DiO dye) was determined by the gain of the DiO dye by NB4 cells (stained with a violet membrane dye) over a time. (B) Representative flow cytometry plots and combined data (C) representing four independent experiments with three technical replicates per datapoint. A paired one‐way ANOVA was used to assess statistical significances.

FIGURE 4

SPPL3 regulates Fc‐protein binding to their ligands on HAP1 WT, SPPL3^−/−, and SPPL3^−/−B3GNT5^−/− cells. HAP1 WT (DiL dye), SPPL3^−/− (Celltrace violet), and SPPL3^−/−B3GNT5^−/− (CFSE) were mixed (A) and incubated with different concentrations of LIR1‐Fc, SIRPα‐Fc, Siglec‐7‐Fc, or NKG2D‐Fc. Representative flow cytometry plots (B) and summarizing graphs for the binding of LIR‐1‐Fc (C), SIRPa‐Fc (D), Siglec‐7‐Fc (E), and NKG2D‐Fc (F). Data represent two (Siglec‐7‐fc and NKG2D‐fc) or three (LIR‐1‐fc and SIRPα‐fc) with two technical replicates per datapoint. A paired one‐way ANOVA was used to assess statistical significances. MFI, mean fluorescence intensity.

FIGURE 5

Antibody binding to proteins on HAP1 SPPL3^−/− and HAP1 SPPL3^−/−B3GNT5^−/− relative to HAP1 WT cells. HAP1 WT (Celltrace violet), SPPL3^−/− (AF350 dye) and SPPL3^−/−B3GNT5^−/− (Celltrace violet and AF350 dye) (A) were mixed and incubated with 168 unique, primary antibodies targeting 34 cell surface proteins. Antibodies targeting a total of 24 proteins passed quality control. (B) A proportion of proteins were targeted with antibodies that were all not affected to bind to their epitopes with SPPL3^−/−, whereas some proteins were partially (at least one of the tested antibodies) or completely affected (all tested antibodies), which could be alleviated with additional B3GNT5^−/−. The ratio of the MFI of each antibody for the SPPL3^−/−, B3GNT5^−/−, and SPPL3^−/−B3GNT5^−/− compared to the WT cells was calculated (example in plots in B). The proteins were grouped on the basis of the effect of the loss of SPPL3 or B3GNT5; proteins with epitopes affected by the loss of SPPL3 (C), proteins of which at least one antibody is not affected by the loss of SPPL3 (D), proteins with epitopes that are not affected by the loss of SPPL3 (E), proteins with epitopes affected by the loss of B3GNT5 or SPPL3 and B3GNT5 (F). The colored bar represents the area that is considered similar to WT (cutoff for difference: >0.8 and <1.2). Data represent two independent experiments and three technical replicates per datapoint. MFI, mean fluorescence intensity.

FIGURE 6

The protrusion of proteins affected by nsGSLs versus unaffected proteins. Available protein structures were obtained from the Alphafold database for the proteins that contained epitopes that were affected (Figure 5C,D) or not affected by nsGSLs (Figure 5E). The distance between the alpha carbons of the first amino acid of the extracellular domain to all other amino acids was determined in Armstrong (Å) using PyMOL, and the longest theoretical distance was selected. For example, (A) the maximum distance of CD9 is 40.5 Å. The rest of the structures are shown in Figure S4. (B) The maximum protruding distance (size) of proteins with versus the proteins without identified affected epitopes. ECD, extracellular domain.

FIGURE 7

The binding of multiple anti‐CD147 antibodies with different affinities to HAP1 WT, HAP1 SPPL3^−/−, HAP1 B3GNT5^−/−, and HAP1 SPPL3^−/−/B3GNT5^−/− cells. Thirteen different antibodies targeting CD147 were incubated with “barcoded” and mixed HAP1 WT (AF350 dye), SPPL3^−/− (Celltrace violet), B3GNT5^−/− (unstained), and SPPL3^−/−/B3GNT5^−/− (CFSE) cells. (A) Representative flow cytometry plots for each antibody. (B) The correlation plot of the degree of SPPL3 sensitivity of binding for each antibody (SPPL3^−/− MFI/WT MFI) versus the affinity, as described by Koch et al., of the antibodies for their epitope 24. (C) Categorization of the epitopes targeted by the antibodies based on the position of the epitopes relative to the cell membrane. Data shown are representative for two independent experiments with three technical replicates per datapoint. MFI, mean fluorescence intensity.