Targeting innate immune pathways to instruct therapeutic immune tolerance

Introduction:: Autoimmune diseases like multiple sclerosis (MS), type 1 diabetes, and lupus occur when the immune system attacks normal host molecules, termed self-antigens. There is no cure for these autoimmune diseases and current therapeutic options can leave patients immunocompromised and susceptible to life-threatening infections. To counter the autoimmune attack, a transformative goal is the development of immunotherapies that promote antigen-specific tolerance. These therapies would impact patient health and quality-of-life by providing effective and durable protection from disease, while preserving healthy immune function. To pursue this impactful goal, we focused on signalling pathways and therapeutic delivery systems to control innate immunity. One exciting target to promote antigen-specific tolerance is toll-like receptors (TLRs); TLRs are innate signaling pathways traditionally studied as the first line of defense against invading pathogens. Remarkably, increased TLR signaling has been observed in autoimmune diseases like MS, which is the focus of the project herein. Thus, controlling these crucial immune pathways presents a design opportunity for MS therapeutics to inhibit downstream inflammation and promote tolerance. Additionally, advances in nanotechnology enable precise assembly of vaccines and immunotherapies. We have utilized a nanoscale assembly platform to build immunotherapies and test the controlled delivery of defined therapeutic formulations and test how their biophysical assembly impacts therapeutic function.


Materials and Methods:: We built a library of myelin self-antigen (myelin oligodendrocyte glycoprotein, MOG) modifications with altered cationic changes by adding arginine (R₂, R₃, R₉) and lysine (K₂, K₃, K₉) residues, where the subscript represents the added number of residues. Affinity between these MOG sequences and an antagonist for neucleotide-sensing TLR9 (GpG) was measured using surface plasmon resonance (SPR). Nanoconstructs assembled from these components were used to treat dendritic cells to analyzed the impact on gene expression. We hypothesized that this modular nanoscale-assembly of therapeutics could be also used to target TLR9 while co-deliverying distinct myelin self-antigens to counter specific models of autoimmunity. We tested this hypothesis in multiple pre-clinical models of MS, experimental autoimmune encephalomyelitis (EAE) and relapsing-remitting EAE (RR-EAE). EAE models the progressive form MS and is driven by an immune attack against the MOG antigen. RR-EAE models the most common form of MS where clinical symptoms fluctuate, and is driven by an immune attack against the myelin proteolipid protein (PLP) antigen. Following treatment we analyzed the impact of healthy immunity by administering a vaccine for an exogenous antigen, then analyze antibodies (ELISA) and antigen-specific T cells (tetramer flow cytometry). Additionally, we utilized the library of therapeutic nanoconstructs to investigate the mechanism-of-action for therapeutic immune tolerance. Statistical calculations were performed in JMP Pro, with all tests and sample sizes being indicated. RR-EAE clinical score data was analyzed with a nonparametric Steel−Dwass test to account for multiple comparisons and data normality.


Results, Conclusions, and Discussions:: Our data shows that electrostatics are a versatile parameter for controlling the self-assembly of immunotherapies and regulating how they function. First, we show using SPR analysis that the affinity between MOG and GpG could be tuned by addition of arginine and lysine residues (Figure 1A). Notably, the formulations with higher affinity between components significantly impacted GpG regulatory function in activated DCs. This is shown in gene expression heatmap, where unsupervised clustering grouped higher Il6, Tnf, and Myd88 gene expression of MOGR₉/GpG and MOGK₉/GpG closer to the positive inflammatory control (Media with CpG stimulation) (Figure 1B). Excitingly, our data has shown that MOG/GpG nanoconstructs reversed established paralysis in the EAE model, while leaving healthy immunity intact. Mice developed antibody and cell-mediated immunity from a vaccine that was administered following nanoconstruct treatment; translating this retention of healthy immunity to the clinic could profoundly improve MS patient health and quality-of-life. To extend these findings to the relapsing-remitting model of MS, we built nanoconstructs form PLP and GpG then treated with either a single dose (peak disease) or two doses (peak and remission). Level of paralysis, as indicated by clinical score, shows that administration of two doses provides durable protection from paralysis during disease relapses (Figure 1C). Interestingly, the single treatment provided a trending level of protection only during the relapses (unpublished data). Using the EAE model and modular assembly of therapeutics, we also show the relative importance of self-antigen and the regulatory cue for promoting therapeutic immune tolerance. To build on this efficacy data, we used high-throughput gene expression analysis of the treatment draining lymph nodes shows that MOG/GpG nanoconstruct delivery elicits a unique immunological profile associated with therapeutic immune tolerance, which is distinct from naïve, soluble controls, or disease. This interesting finding highlights the impact of biophysical presentation of signals and provides insight on the modulation of innate immune signaling to drive therapeutic immune tolerance. Collectively, this data shows that targeting innate immune pathways and cells promotes therapeutic immune tolerance and counters autoimmunity.


Acknowledgements (Optional): : Supported by VA Merit Award I01BX003690 (Jewell), NIH R01EB027143 (Jewell), VA Career Development Award IK2BX005061 (Oakes), and NSF GRFP DGE 1840340 (Froimchuk).


References (Optional): : Oakes et al., ACS Nano. 2021; 15, 4305–4320. Froimchuk et al., Nano Letters. 2021; 21, 3762-3771.