Immunity. 2020 Nov 25;S1074-7613(20)30468-4.
doi: 10.1016/j.immuni.2020.11.003.
Background：
Over the last 35 years, several key elements in HLA-I expression and antigen presentation have been identified and extensively studied. A protein complex directed by the transcriptional regulator NLRC5 drives HLA-I expression in selected tissues, whereas HLA-I translation and glycosylation are thought to be executed by general enzymes and mechanisms. In the ER, the HLA-I heavy chain and its light chain beta-2 microglobulin (β2m) assemble and are stabilized by a unique combination of the ER chaperone proteins: tapasin (TAPBP), ERp57 (PDIA3), and calreticulin (CALR). These HLA-I chaperone complexes bind the peptide transporter TAP to form the peptide-loading complex (PLC), which drives efficient ER import and loading of peptides into the HLA-I peptide binding groove. Subsequently, mature trimeric complexes of HLA-I heavy chain, b2 m, and peptide are released from the PLC and transported to the cell surface for peptide presentation to T cells. Given the multifactorial complexity of the HLA-I antigen presentation pathway, the authors hypothesized that additional regulatory mechanisms of this central process in adaptive immunity must exist.
Methods:
Haploid genetic screening;BFA (Brefeldin A) assay: to determine HLA-I turnover;Reverse-phase (RP) nanoLC-ESI-MS(/MS);Single particle tracking of HLA-I molecules: cells were stained with a single molecule dilution of the BB7.2-Fab-AF555
Results:
1. To identify unknown factors regulating HLA-I antigen presentation, the authors performed a genome-wide insertional mutagenesis screen in haploid human fibroblast-like HAP1 cells endogenously expressing HLA-I. Subsequently, they determined the relative enrichment of unique disruptive integrations per gene between the sorted populations using deep sequencing. A haploid genetic screen reveals SPPL3 (an ER- and Golgi-localized transmembrane protein of the family of intramembrane-cleaving aspartyl proteases) as a regulator of antibody accessibility to membrane-proximal HLA-I regions.2. To validate that HLA-I cell surface expression was altered by SPPL3, the authors created SPPL3-/- HAP1 cells using CRISPR-Cas9. HLA-I surface expression in the absence of SPPL3 turned out identical to those of WT cells. Furthermore, SPPL3 deficiency did not alter HLA-I turnover or stability of the peptide-HLA-I interaction.3. To test whether these seemingly contradictory outcomes may have resulted from reduced accessibility of the W6/32 epitope in the absence of SPPL3, the authors titrated the W6/32 antibody for binding to WT, SPPL3-/- , and control HLA-A-/- B-/- C-/- (HLA-I-deficient) and TAPBP-/-cells. In contrast to saturating W6/32 concentrations, lower W6/32 concentrations resulted in decreased binding to SPPL3-/- cells compared with WT cells, indicating that accessibility of the HLA-I epitope recognized by W6/32 was indeed hindered.4. By superimposing critical amino acid positions for binding of the individual antibodies onto an HLA-I structure, the authors defined the SPPL3-susceptible region as relatively proximal to the cellular membrane, an area that is largely conserved among HLA-I alleles.5. To test whether the HLA-I function is affected by SPPL3, the authors evaluated whether SPPL3 enhanced HLA-I antigen presentation to CD8+ T cells by stimulating multiple HLA-A*02:01-restricted T cell clones specific for different tumor-expressed antigens endogenously expressed by WT and SPPL3-/-cells. All clones were more reactive to SPPL3-expressing WT cells, as determined by their IFN-g or granulocyte-macrophage colony-stimulating factor (GM-CSF) production.6. Even more pronounced than for SPPL3-affected antibodies, binding of a recombinant LIR-1 Fc fusion protein to HLA-I was strongly decreased on SPPL3-/-cells compared with WT cells, suggesting that various immune cell functions can be affected by SPPL3.7. To investigate whether SPPL3 catalytic activity was critical in controlling protein accessibility to HLA-I and function, the authors expressed WT SPPL3 or a catalytically inactive SPPL3 mutant (D271A) in SPPL3-/- cells. Flow cytometry analysis showed that only SPPL3 D271A failed to restore the accessibility of HLA-I for W6/32, indicating that SPPL3 proteolytic activity is required for antibody accessibility to HLA-I.8. To searche for potential genes negatively regulated by SPPL3 to affect HLA-I the authors performed a genome-wide haploid screen in SPPL3-/- HAP1 cells. The results revealed the existence of apathway comprising GSL-mediated regulation of HLA-I access and function controlled by SPPL3.9. To validate that SPPL3 reduces protein accessibility to HLA-I through manipulation of GSL synthesis, the authors generated GSL-deficient SPPL3-/-cells by additional genetic ablation of the first enzyme of the GSL synthesis pathway, UGCG. In these SPPL3-/- UGCG-/- cells, full rescue of the W6/32 HLA-I epitope accessibility without affecting SPPL3-independent B1.23.2 staining was observed, pointing toward an essential role for GSLs in shielding specific HLA-I epitopes.10. The screening data suggested that lacto-series GSL or nsGSL (neolacto-series) production, through B3GNT5 activity, could diminish protein accessibility to HLA-I. And the authors confirmed this specificity by generating polyclonal celllines on the SPPL3-/- background, each CRISPR-Cas9 targeted for one of the five branching enzymes, and then analyzed W6/32 binding by flow cytometry.11. To detect a direct interaction between SPPL3 and its putative target B3GNT5, the authors performed coimmunoprecipitation of over-expressed epitope-tagged proteins. It turned out that B3GNT5 is a specific substrate of SPPL3.12. To investigate whether SPPL3 affects B3GNT5 activity, the authors performed a B3GNT5 enzymatic assay. The B3GNT5 product lactotriaosylceramide (BODIPY-Lc3Cer), as confirmed by LC-MS, was generated in increased amounts in SPPL3-/- compared with WT cell lysates.13. The authors then investigated the mobility of HLA-I in SPPL3-/- cells by single-particle tracking. The mobile fraction and diffusion constant of BB7.2 Fab labeled HLA-I molecules were equal between SPPL3-/- and WT cells.14. Further analyses of the GSL signature of SPPL3-/- compared with WT cells revealed that the nsGSL glycan chains more frequently contain a-2,3- and a-2,6-linked sialic acid residues, as well as noncharged fucoses. And the enzymatic removal of sialic acid residues at the cell surface by neuraminidase treatment diminished HLA-I shielding.15. After determining that nsGSL-rich target cells suppress T cell activity, the authors examined the effect of increased nsGSL expression or downmodulated SPPL3 activity intumors. Elevated amounts of nsGSLs or their synthesis enzyme B3GNT5 have been observed on several tumor types, including glioma, acute myeloid leukemia (AML), and adenocarcinomas. And pharmacological inhibition of GSL Synthesis in glioma enhances anti-tumor immune activation in vitro.
Conclusions:
Iterative KO screens reveal a new pathway controlling HLA-I antigen presentation.SPPL3 suppresses B3GNT5 activity affecting the cell surface GSL repertoire.B3GNT5-generated GSLs limit the capacity of HLA-I to interact with natural ligands.Inhibition of GSL synthesis in glioma enhances anti-tumor immune activation.