Insights into the CD1 lipidome

Abstract

CD1 isoforms are MHC class I-like molecules that present lipid-antigens to T cells and have been associated with a variety of immune responses. The lipid repertoire bound and presented by the four CD1 isoforms may be influenced by factors such as the cellular lipidome, subcellular microenvironment, and the properties of the binding pocket. In this study, by shotgun mass spectrometry, we performed a comprehensive lipidomic analysis of soluble CD1 molecules. We identified 1040 lipids, of which 293 were present in all isoforms. Comparative analysis revealed that the isoforms bind almost any cellular lipid.CD1a and CD1c closely mirrored the cellular lipidome, while CD1b and CD1d showed a preference for sphingolipids. Each CD1 isoform was found to have unique lipid species, suggesting some distinct roles in lipid presentation and immune responses. These findings contribute to our understanding of the role of CD1 system in immunity and could have implications for the development of lipid-based therapeutics.

Keywords: CD1; antigen presentation; lipid mass-spectrometry; lipidome; self-lipid antigens.

Figure 1

Lipidome of CD1 isoforms (A) Principal Component Analysis of the lipid classes bound to each CD1 isoform. (B) Venn diagram of overlapping lipid classes between the CD1 isoforms. The diagram shows the number events detected in each isoform.

Figure 2

Shared cellular lipids captured by the CD1 isoforms. Mol percentage of the 293F shared lipid classes captured by the indicated CD1 isoforms in comparison with the 293F cellular lipidome. The graphs show only the proportion of lipids that are present in all isoforms. The identified lipid molecules were quantified by normalization to a lipid class-specific internal standard. The amounts in pmoles of individual lipid molecules (species of subspecies) of a given lipid class were summed to yield the total amount of the lipid class. The amounts of the lipid classes were then normalized to the total lipid amount yielding mol% per total lipids. Ceramide (Cer), Diacylglycerol (DAG), Monosialodihexosylganglioside (GM3), hexosylceramide (HexCer), lyso-phosphatidyl-cholines (LPC), ether-linked lyso-phosphatidyl-ethanolamine (LPE O-), phosphatidylcholine (PC), ether-linked Phosphatidylcholine (PC O-), phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE O-), phosphatidylglycerol (PG), sphingomyelin (SM), Sulfatide (Sulf).

Figure 3

Over capture analysis of the shared CD1 lipidomes. Mol percentage of the nine most abundant shared lipid classes eluted from CD1 molecules, compared to the cellular background (dashed line). (A-D) Phospholipids. (E-G) Headed sphingolipids. (H, I) Headless lipids. Ceramides showed strong capture by CD1d but lower capture by other isoforms. SMs were over-captured by CD1b, CD1c and CD1d. General pattern of under-capture of phospholipids (PE, EPE, and EPC). GM3 was over-captured by CD1b, CD1c and CD1d.

Figure 4

Mol% of unique lipids of the CD1 isoforms. The CD1a unique features were PI and PS; the CD1b lipidome was enriched in PG and sulfatide; the CD1c-specific lipidome was enriched in GM2 (36:1;2), GD1, and GD2 species; Cholesterol was enriched in the CD1d unique lipidome.

Figure 5

Chain length profile of the lipids captured by the CD1 isoforms. Lipid chain length captured in the shared CD1 lipidome expressed as a mol percentage of sample.

Figure 6

Total length of unique lipids captured by the CD1 isoforms. Lipid chain length captured in the CD1 isoform-specific lipidome expressed as a mol percentage of sample.

Figure 7

Abundance analysis of PC and PC O- species across CD1 isoforms. The bubble size indicates the abundance of the lipid species in each isoforms lipidome expressed as z-score (describing the positive or negative deviance from the mean in standard deviation units). CD1a bound PC chains of middle length (C38–C40); CD1b captured the shortest chain length of PC (C30–C38); CD1d skewed towards long-chain PCs (C42– C46); CD1c bound to the full spectrum of PC species (C30–C46).

Figure 8

Abundance analysis of selected Sphingolipids in CD1 isoforms. The bubble size indicates the abundance of the lipid species in each isoforms’ lipidome expressed as z-score. CD1a bound to larger SM species (C40-C42); CD1b captured the shortest chain length of SMs (C34); CD1c bound to C34-C36 SMs and CD1d didn’t show any SM chain length preference. GM3 was most abundantly captured by CD1d.