Investigating the differential effects of LILRB1 natural variants on interaction with MHC-I and UL18

• Author / Creator

  Azaizeh, Bara'ah

• The Leukocyte immunoglobulin-like receptor B1 (LILRB1) is widely expressed throughout human immune cells of lymphoid and myeloid origins. It is also associated with viral infection, autoimmune diseases and cancer. LILRB1 regulates immune responses through broad interaction with major histocompatibility complex class I (MHC-I) molecules and UL18, an immune-evasion protein of human cytomegalovirus (HCMV). The first two immunoglobulin (Ig) domains (D1D2) of LILRB1 engage the highly conserved α3 and β2m domains of MHC-I and UL18. Our group previously studied three variants and showed polymorphisms in LILRB1 outside the contact site with MHC-I influence the interaction with MHC-I as well as UL18. However, the four non-synonymous SNPs situated in the coding region of D1D2 give rise to seven LILRB1 protein variants.This thesis aimed to expand the characterization of the polymorphic residues to the full set of the seven variants in terms of binding with MHC-I and UL18, effects on receptor Fc-protein protein production, and cell surface expression of the full-length receptor. By generating a soluble form of the D1D2 domains tagged with Fc for use in a flow cytometry-based binding assay, I found that polymorphism of residue 119 that controls the addition of an N-linked glycan to the D2 domain has a significant impact on secretion of the D1D2-Fc constructs. Interestingly, the effect of polymorphisms on cell surface expression of the intact receptor indicated higher expression of variants with proline at residue 45. Further investigation of the very low expression of variants with a leucine substitution at the same position points to possible involvement of post translation mechanisms unique to this polymorphism. Next, I examined binding using the soluble D1D2-Fc fusion proteins to cell surface MHC-I and a reporter cell system with the full-length extracellular domains. The Fc-fusion proteins of the seven different D1D2 domains display a range of binding strengths with the classical MHC-I proteins HLA-B58 and HLA-Cw15. The variants’ ability to stimulate reporter cells expressing all extracellular domains (D1-D4) of LILRB1 on the cell surface, were similar to the binding pattern of the Fc-proteins. In contrast, I observed the interaction with HLA-G is indistinguishable for the majority of the LILRB1 variants (5 out of 7). For the two variants that deviate from the group, one has a significantly stronger and the other a much weaker interaction with HLA-G. In a similar way I also characterized the effects of LILRB1 polymorphisms on UL18 interaction considering different UL18 mutations. In the process to obtaining sufficient expression of UL18 derived from three different strains, I described a variation of surface expression among the three UL18’s of AD169, Davis and Towne. Such variation between strains is likely due unique mutations in each strain, where mutation of a shared α3 located sequence (DVE) into a NAD is associated with marked decrease of surface expression. Furthermore, the abrogation of two conserved retention motifs in the UL18 cytoplasmic tail, led to general enhancement of surface expression by most UL18 regardless of other mutations throughout the heavy chain. The UL18 cytoplasmic mutants were used to conduct the binding assays with LILRB1 variants which yielded similar binding between UL18 with DVE or NAD sequence. However, consistent with our group’s previous finding, one LILRB1 variant displayed strong binding with AD169 and Towne compared to other variants. Also, several other variants displayed variation in binding with Towne at high concentrations suggesting an influence of the UL18 sequence. To my knowledge this is the first research examining LILRB1 variants interaction with HLA-G, the effect of NAD mutation of UL18 surface expression and interaction with the different LILRB1 variants. This research supports the possible influence of UL18 mutation on both surface expression and LILRB1 binding. Furthermore, the high degree of conservation of the binding strength with HLA-G relative to classical MHC-I and UL18 supports the notion that, while pathogens may drive the diversification of LILRB1, consistent interaction with HLA-G is important for alleles to be retained in the population.

• Subjects / Keywords

  • LILRB1
  • MHC-I
  • UL18

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-1r3h-9z03

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.