(M1430-02-08) Structural Modifications of Phosphatidylserine and Their Impact on Oral Tolerance

Purpose: Immunogenicity against therapeutic proteins, autoimmunity and food allergy, are caused by unwanted immune responses against protein antigens. To prevent these unwanted immune responses, we have developed phosphatidylserine (PS) containing lipid nanoparticles that mimic the surface properties of apoptotic cells, converting an immunogen into a tolerogen. Previous studies have shown that mice prophylactically treated with PS nanoparticles—either double-chain PS or single-chain PS (LysoPS), administered intravenously or subcutaneously, suppressed humoral immune responses against therapeutic proteins such as Factor VIII (FVIII) or α-Glucosidase (GAA). However, the tolerogenic effects observed with oral administration were found to depend on the structure of PS. Mice pre-treated with double-chain PS failed to induce oral tolerance, whereas single-chain LysoPS successfully induced oral tolerance. This difference in the tolerance-inducing potential of PS nanoparticles clearly suggests the relationship between PS structure and oral tolerance. Therefore, this study aims to investigate the structure-function relationship of PS in oral tolerance.

Methods: Ovalbumin (OVA) was used as a model antigen to understand the mechanism of oral tolerance mediated by PS. We used PS that varied in acyl-chain number, degree of unsaturation, and carbon chain length and it was mixed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at 30%:70% mole ratio. The lipid nanoparticles were formed using thin film dehydration and rehydration method, followed by high-pressure membrane extrusion, to generate uniform and homogeneous nanoparticles with a diameter of 100 nm. To investigate the tolerogenic potential toward OVA antigen using PS nanoparticles, we orally immunized Swiss Webster mice continuously for 9 weeks. Starting in week 5, mice were rechallenged with 2 ug of OVA protein subcutaneously for 4 weeks and study was completed by week 11. Blood samples were collected to measure anti-OVA antibodies. Spleen and bone marrow were harvested to assess frequency of Tregs and plasma cells. In week 5, before the subcutaneously OVA rechallenge, we also collected mesenteric lymph nodes (MLN) and conducted single cells RNA sequencing (ScRNA-seq).

Results: The data showed that PS structure influenced its exposure on the nanoparticle surface, which in turn influenced oral tolerance towards OVA. The high PS exposure was associated with lower OVA specific antibody titers, a corresponding reduction in plasma cells and an increase in LAP+ regulatory T-cells. LysoPS nanoparticles containing 18:1 provided optimal oral tolerance with the highest PS exposure on nanoparticle surface. Single-cell RNA Sequencing (ScRNA-seq) reveals that LysoPS 18:1 nanoparticles increased the expression of RNAs associated with tolerogenic features in B-, T-, NKT cells and dendritic cells, particularly through TGF-β responses. Specifically, T-cells received tolerogenic signals from dendritic cells, B and NKT cells.

Conclusion: The structure of PS plays a critical role in oral tolerance. The PS structural changes alter biophysical characteristics of the nanoparticle such as PS exposure and surface charge density, which in turn influences tolerance mechanism. This structure-function relationship has guided the development of an effective oral immunotherapy to treat clinical conditions caused by unwanted immune responses.

References: Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654.
Nguyen, N.H., et al., Rational design of a nanoparticle platform for oral prophylactic immunotherapy to prevent immunogenicity of therapeutic proteins. Sci Rep, 2021. 11(1): p. 17853.
Glassman, F.Y., et al., Phosphatidylserine Is Not Just a Cleanup Crew but Also a Well-Meaning Teacher. J Pharm Sci, 2018. 107(8): p. 2048-2054.

Acknowledgements: Funding was provided by the National Institute of Allergy & Infectious Disease RO1 AI169296 to SVB. The authors would like to acknowledge the UB Optical Imaging and Analysis Facility for the use of the flow cytometer and confocal fluorescence imaging and UB Genomics and Bioinformatics Core for ScRNA-seq


Impact of PS structure on humoral response, plasma cell counts and Treg. (A) PS exposure in various PS nanoparticles. (B) Frequency of CD4+ Foxp3- Lap+ (Th3) Tregs in the spleen. (C) Frequency of bone marrow derived B220+CD138+ plasma cells. (D) OVA-specific IgG1 antibody was measured using indirect ELISA. Data are shown as mean ± SD. Statistical significance was denoted by P < 0.05 (*). The statistical analysis was performed using one-way ANOVA followed by Tukey post-hoc analysis.


Figure 2. LysoPS nanoparticle-induced tolerogenic signatures in the MLN A. The dot plot represents gene expression in the datasets according to treatment groups. The size of the dot represents the percentage of gene expression in the cell population, and the color represents the level of gene expression in the cell population. White indicates a lower expression, as the expression increases, it labels as gray and then black. B. Chord plot showing cellular interaction between cell types involved in TGF-β signaling pathways.