Phosphatidylserine-mediated Oral Tolerance

Abstract

Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and silently clear these dying cells to maintain tolerance via immunological ignorance. However, recent and emerging evidence has demonstrated that PS converts an “immunogen” into a “tolerogen”, and PS exposure on the surface of cells or vesicles actively promotes a tolerogenic environment. This tolerogenic property depends on the biophysical characteristics of PS-containing vesicles, including PS density on the particle surface to effectively engage tolerogenic receptors, such as TIM-4, which is exclusively expressed on the surface of antigen-presenting cells. We harnessed the cellular and molecular mechanistic insight of PS-mediated immune regulation to design an effective oral tolerance approach. This immunotherapy has been shown to prevent/reduce immune response against life-saving protein-based therapies, food allergens, autoantigens, and the antigenic viral capsid peptide commonly used in gene therapy, suggesting a broad spectrum of potential clinical applications. Given the good safety profile of PS together with the ease of administration, oral tolerance achieved with PS-based nanoparticles has a very promising therapeutic impact.

Introduction - Phosphatidylserine

Phosphatidylserine (PS) is an anionic phospholipid expressed abundantly on the eukaryotic plasma cell membrane and plays multiple important roles in biological processes, including thrombin generation, apoptosis, complement system activation, and virus entry.[1] PS is most commonly known for its role in apoptosis, which is a programmed biological process to eliminate unwanted, aged, or damaged cells in a non-inflammatory manner to maintain tissue homeostasis. The role of PS in apoptosis was first described and demonstrated on apoptotic lymphocytes, in which the exposure of PS-expressing apoptotic lymphocytes to macrophages triggered engulfment and removal of the dying lymphocytes.[2] PS is a major component of the cell membrane and, under normal conditions, is primarily restricted to the inner leaflet of eukaryotic cell membrane by the ATP-dependent flippase enzyme.[3] When cells undergo apoptosis, caspases and scramblase expressed on the cell membrane will disrupt lipid asymmetry and rapidly translocate PS from the inner leaflet to the outer surface, leading to the generation of several immunological ignorance signals, including the “eat-me”, and “ignore me” signals to maintain tolerance towards self-proteins and dying cells. The “find-me” signal is a chemotactic response to attract and recruit phagocytic cells, such as macrophages and dendritic cells (DCs), to the environment. Several molecules on apoptotic cells that were also shown to initiate the “find-me” signal include lysophosphatidylcholine and sphingosine-1-phosphate.[4] The translocation of PS from the inner leaflet of cell membrane to the outer surface serves as the essential “eat-me” signal for phagocytes to recognize apoptotic cells.[5] Following the endocytosis of PS-expressing cells, the release of anti-inflammatory cytokines such as TGF-β and IL-10 provides the “ignore me” signal to maintain a non-inflammatory environment.[6, 7] These unique properties of PS not only serve in the mechanism of programmed cell death but are also adapted as a defensive and invasive mechanism by tumor cells, viruses, and parasitic organisms to evade the host’s immune system.[6]

PS Is Not a Clean-up Crew but a Well-meaning Teacher

Although apoptosis is traditionally regarded as a silent event to clear apoptotic cells without the initiation of immune responses to maintain tissue homeostasis, recent evidence from our group demonstrated that PS-mediated cellular uptake is an active teaching process to induce peripheral tolerance towards self and foreign antigens.[8–12] The use of nanoparticles made of PS (containing a serine head group connected to two fatty acyl chains, referred to as double-chain PS) containing antigens/proteins given via parenteral routes of administration, to achieve immune tolerance has been demonstrated in our laboratory. The antigens used include different therapeutic proteins and autoantigens such as factor VIII (FVIII), recombinant human acid alpha-glucosidase (rhGAA), and myelin oligodendrocyte glycoprotein peptide (MOG_35–55), in gene-knockout and disease-induced mouse models. For example, the immunogenicity of FVIII and the potential of PS to induce tolerance were evaluated in Hemophilia A (HA) mice, as the development of anti-FVIII neutralizing antibody in 30% of HA patients is a major clinical complication of this bleeding disorder.[9] Animals were pre-treated with either free FVIII, FVIII in combination with dexamethasone, a general immunosuppressant, or FVIII associated with PS nanoparticles (PS-FVIII) via the subcutaneous (SC) route, followed by four weekly rechallenge injections with FVIII alone. At the end of the study, mice pre-treated with PS-FVIII displayed significantly low anti-FVIII titer levels despite the rechallenge injections. In sharp contrast, mice receiving FVIII alone or FVIII in combination with dexamethasone developed high and comparable anti-FVIII antibodies. This indicates that the immune systems of animals receiving dexamethasone responded normally to FVIII when the pre-treatment with dexamethasone stopped and animals were rechallenged with free antigens. On the other hand, PS-FVIII induces immunological hypo-responsiveness toward FVIII even after the PS treatment stopped. When HA mice were concomitantly given ovalbumin (OVA) and PS-FVIII at distant anatomical sites, the immune system responded normally to OVA, but tolerance towards FVIII was still maintained.[9] Therefore, the tolerogenic effect of PS is antigen-specific rather than a systemic immune suppression. Using a similar experimental design, Glassman et al. also demonstrated that pre-exposure of mice homozygous for the disruption of acid alpha-glucosidase gene, a murine model for Pompe disease, to rhGAA in the presence of PS nanoparticles reduced/prevented immune responses to rhGAA despite the subsequent re-administrations with free rhGAA.[8] Meanwhile, treatment of rhGAA in combination with dexamethasone failed to subdue the immune responses following rhGAA re-administrations. The “teaching function” of PS to convert an immunogen to a tolerogen was later extended to reduce autoimmunity against MOG_35–55 peptide in mice having experimental autoimmune encephalomyelitis (EAE), a demyelinating disease mouse model that resembles multiple sclerosis in human.[8] In this experiment, mice received PS-MOG_35–55 prior to EAE disease induction displayed a significant delay in disease onset with significantly low cumulative disease scores, indicating lower disease severity, compared to animals receiving buffer or MOG_35–55 alone.

Further investigations on the mechanism of PS-mediated immune tolerance suggest the involvement of regulatory T cells (T_regs), as the adoptive transfer of CD4⁺CD25⁺ activated T cells from PS-FVIII treated mice to naïve untreated animals led to a significant reduction in anti-FVIII titers in recipient mice, despite a series of FVIII rechallenge injections in these mice.[9] In contrast, animals receiving activated T cells from donor mice treated with FVIII alone developed a robust immune response against FVIII. This suggests that the tolerogenic effect of PS is transferable, and further evaluation of the T cell phenotype revealed the activity of PS-induced Foxp3⁺ T_regs.

PS Receptors in Immune Regulation

Several PS receptors on the surface of phagocytic cells responsible for PS recognition, binding, and subsequent cellular signaling have been identified. Among these receptors, transmembrane immunoglobulin and mucin domain (TIM) receptor and trio receptors Tyro3, Axl, and Mer (TAM) are shown to participate in PS-mediated immune regulation. TAM receptors are primarily expressed on the surface of antigen-presenting cells (APCs) such as macrophages, DCs, and natural killer cells.[13] The roles of TAM in PS signaling leading to anti-inflammatory processes have been described. [6, 14, 15] Besides TAM, TIM is a transmembrane protein that binds to PS to facilitate apoptotic cell engulfment and a broad range of other immune responses. Among members of the TIM family, TIM-4 was shown to play a critical role in PS-mediated tolerance induction. Particularly, when HA mice were pre-exposed to FVIII in the presence of PS nanoparticles (PS-FVIII), a significant reduction of anti-FVIII titer levels was observed.[8] However, the administration of the function-blocking anti-TIM-4 antibody prior to treatment with PS-FVIII significantly abrogated the tolerance-induction effect of PS. Therefore, TIM-4 receptor on the surface of APCs is essential for PS-mediated immune tolerance, and vesicles expressing surface PS bind to TIM-4 in a PS-density-dependent manner.[16] Moreover, PS receptors play a critical role in tolerance versus silent clearance of PS-exposing cell debris and vesicles, and the structural/biophysical characteristics of PS vesicles can drive the immune response type (i.e. tolerance versus ignorance).

PS-mediated Oral Tolerance

The use of nanoparticles containing double-chain PS have been successful in reducing immune responses when given via parenteral route of administration. However, to further optimize the tolerogenic potential of this nanoparticle platform and to expand its clinical utilities to provide ease of administration to a broader range of patients, including pediatrics, several approaches were investigated, including switching to the oral route of administration and modifying the structural properties of PS-containing nanoparticles.

1. Structural and Biophysical Characteristics of PS-based Nanoparticles

While the common structure of PS primarily consists of a serine headgroup connected to two fatty acid acyl chains by a glycerol molecule (double-chain PS), several derivatives of PS can exist, which differ in the acyl chain length, number of acyl chains, and degree of unsaturation. These structural modifications can influence the biological functions of PS, as not all PS exposure leads to the same biological outcome.[1] Although TIM-4 receptor is essential for PS-mediated tolerance induction,[8] this receptor displays selective and strong binding to cells or particles expressing high PS surface density, as one TIM-4 receptor can interact with multiple PS molecules.[16] Therefore, the rational design of a nanoparticle with high PS surface exposure would facilitate the interaction with TIM-4 and other tolerance-inducing receptors on APCs, leading to more effective immune tolerance induction. Among different PS derivatives, lysophosphatidylserine (Lyso-PS) is a single-acyl-chain version of PS with one degree of unsaturation. Because of the difference in the number of acyl chains, nanoparticles containing single-chain Lyso-PS had a significantly smaller mean hydrodynamic diameter than those comprised of double-chain PS, despite the identical preparation procedures.[17, 18] This is likely due to an increase in membrane surface curvature caused by the cone-shaped Lyso-PS, in combination with the packing defects caused by the arrangement of Lyso-PS bearing kinked unsaturated acyl chain. More importantly, Lyso-PS nanoparticles displayed substantially higher PS surface density than PS nanoparticles, leading to the non-uniform clustering of single-chain Lyso-PS on the surface of the nanoparticles, whereas double-chain PS distributes more uniformly on both the inner and outer layers of the vesicles. [18] The beneficial combination of small particle size and high PS surface exposure of nanoparticles containing Lyso-PS can provide more available total surface area for selective and differential recognition by tolerance-inducing receptors, such as TIM-4, for effective tolerance induction.

2. Oral tolerance

The oral route of administration has been extensively studied for its ability to induce antigen-specific tolerance. Under the high antigen pressure that our guts encounter daily, the intestinal immune system has naturally evolved as a tightly regulated tolerance-induction site and anti-inflammatory environment, which helps tolerate harmless food antigens and commensal bacteria while still protecting the gut from pathogens.[19, 20] By delivering Lyso-PS nanoparticles via the oral route, we aim to target the gut-associated lymphoid tissue (GALT) in the gastrointestinal (GI) tract to induce antigen-specific oral tolerance to any proteins of interest in a user-friendly manner. In the lamina propria (LP), the transport of antigens to the mesenteric lymph nodes (MLNs) by migratory CD103⁺ DCs is crucial for the induction of systemic oral tolerance.[21] CD103⁺ DCs can acquire antigens through several mechanisms: (1) passive phagocytosis of antigens by enterocytes, (2) antigen transport across the gut by M cells on Peyer’s patches or goblet cells, and (3) antigen transfer by CX3CR1^hi macrophages.[22, 23] Following antigen processing, CD103⁺ DCs upregulate the chemokine receptor CCR7 to migrate to MLNs [24], where they induce T_regs proliferation for oral tolerance with the help of cytokines, enzymes, and metabolite signals such as TGF-β, indoleamine 2,3-dioxygenase, and retinoic acid.[25–27]. On the luminal side of the Peyer’s patches, microfold (M) cells are a special type of epithelial cells responsible for the uptake and transport of particulate antigens across the mucosal barrier into GALT.[28–30] Following the phagocytosis and transcytosis via M cells, antigens exit into the intraepithelial pocket, which harbors various populations of lymphocytes and APCs for antigen processing to initiate appropriate mucosal immune responses.[31]

Macrophages and DCs are the major APC populations within the GI tract responsible for antigen sampling and immune homeostasis. At steady state, TIM-4 is expressed primarily by LP macrophages (75% of total macrophages) but not DCs.[32] However, CD103⁺ DCs upregulate the expression of TIM-4 (approximately 30% of total DCs) and co-stimulatory molecules following antigen exposure. They also phagocytose apoptotic cells and soluble antigens more effectively than TIM-4⁻ counterparts. More importantly, TIM-4⁺CD103⁺ DCs also migrate to the MLNs for T cell priming and these DCs likely correspond to the cell subsets identified in previous reports by Jang et al. and Huang et al., which demonstrated that some DC populations in LP constitutively phagocytosed apoptotic intestinal epithelial cells.[33, 34] These DCs then migrated to the MLNs to present antigens associated with the apoptotic cells to naïve T cells for subsequent expansion of T cells producing anti-inflammatory cytokines IL-4 and IL-10. Collectively, these observations imply the immunomodulatory role of intestinal TIM-4⁺ macrophages and DCs, especially CD103⁺ DCs, and that Lyso-PS nanoparticles, which mimic the apoptotic cell construct with favorable biophysical characteristics, can actively engage these professional APCs for oral tolerance induction. Apart from TIM and TAM receptors, PS exposure can engage other PS binding molecules such as annexin V and glycoprotein 2 (GP2). GP2 is an uptake receptor highly expressed on the intestinal M cells for antigen sampling[35] and was shown to contribute to Lyso-PS nanoparticle uptake in the Peyer’s patches.[17] Annexin V is a cellular protein that is highly expressed on M cell surface.[36] The high binding affinity of annexin V to PS and the role of the annexin family in membrane trafficking and endocytosis [37] further suggest the potential role of annexin V in the differential transport of Lyso-PS nanoparticles expressing high PS on the surface. The high expression of scavenger receptor class B type 1, a PS receptor, and clusterin, a lipid transport protein, on M cells [36] also proposes the potential involvement of these molecules in the transport of Lyso-PS nanoparticles, followed by selective and differential interaction of Lyso-PS nanoparticles carrying antigens with TIM-4-expressing APCs located beneath M cells.

Oral Tolerance Induced by Lyso-PS Nanoparticles

1. Differential Uptake of PS-based Nanoparticles following Oral Administration

Because Lyso-PS nanoparticles are stable in the harsh environment of the GI tract, they can be administered orally to target GALT for tolerance induction.[18] Although nanoparticles containing double-chain PS display similar stability in the GI tract following oral administration, single-chain Lyso-PS nanoparticles are transported more efficiently by M cells across the mucosal barrier than double-chain PS nanoparticles.[17] In another follow-up study, in which AF488-labeled OVA encapsulated within rhodamine-labeled nanoparticles were injected into the gut loop, a substantially higher uptake of Lyso-PS nanoparticles carrying OVA was detected than double-chain PS nanoparticles by all immune cell subsets beneath M cells in the Peyer’s patches, especially by CD103⁺ DCs and macrophages (Fig. 1). This reinforces the favorable uptake of Lyso-PS nanoparticles by M cells to the subepithelial dome of the Peyer’s patches, and the active phagocytosis and processing of antigens in the context of Lyso-PS by APCs, predominantly by the tolerogenic CD103⁺ DCs and macrophages, to prime naïve T cells into Tregs for tolerance induction. It is likely that the synergistic effects of small particle size, high PS surface exposure, and the presence of small-scale Lyso-PS-rich domains allow Lyso-PS nanoparticles to be taken up more effectively by M cells.[38]

Figure 1: Uptake of PS and Lyso-PS nanoparticles by different immune cell subsets in the Peyer’s patches.

Twenty μg of OVA-AF488 was loaded into rhodamine-labeled PS and Lyso-PS nanoparticles. One-cm section of the mouse’s intestinal loop containing the Peyer’s patches was surgically ligated and formulations were injected into this loop. The Peyer’s patches were harvested 1 h post-injection, digested, and stained with viability dye, B220, CD3, F4/80, CD11c, and CD103 antibodies prior to flow cytometry analysis. Data were presented as the frequency of cells double-positive for OVA and nanoparticle signal at respective cell populations (mean ± SD). Statistical analysis was performed using a one-way ANOVA test.

Consistent with this observation, the ability of nanoparticles containing the single-chain Lyso-PS, but not double-chain PS (although both induced tolerance when administered via SC and IV routes), to induce antigen-specific oral tolerance has been established with multiple antigens in both general and gene-knockout disease mouse models. Furthermore, this highlights that the biophysical characteristics and design of nanoparticles are critical for tolerance induction when given via different routes of administration. These experiments are discussed in detail below.

2. Oral Tolerance Induced by Lyso-PS Nanoparticles to Prevent Immunogenicity of Therapeutic Proteins

2.1. Oral Tolerance towards FVIII in HA Mouse Model

The oral tolerance induction potential of Lyso-PS nanoparticles was first observed with recombinant FVIII used in the treatment of HA, which is an inherited bleeding disorder caused by the lack of functional FVIII production. About 30% of the patients with severe HA develop inhibitory anti-FVIII antibodies, referred to as inhibitors, against the first-line treatment with recombinant FVIII.[39–42] In patients with high responding inhibitory titers, effective clinical options often become limited, expensive, and ineffective in many cases.[43] When HA mice were continuously pre-exposed to Lyso-PS-FVIII via oral gavage, FVIII inhibitors were undetectable in 75% of the animals despite a series of rechallenge injections with FVIII alone.[18] A similar outcome was later observed with another long-acting FVIII modality: FVIII Fc fusion protein.[44] This suggests that FVIII associated with Lyso-PS nanoparticles is a tolerogenic form of FVIII that can prevent inhibitor development. In contrast, our previously investigated double-chain PS nanoparticles, which induced antigen-specific hyporesponsiveness following IV and SC administrations, were not effective in inducing oral tolerance. Instead, mice receiving oral PS-FVIII elicited titers that were comparable to those receiving buffer and free FVIII.[18] This is consistent with the observation that PS surface exposure of Lyso-PS nanoparticles is significantly higher than that of double-chain PS, leading to differential uptake and transport of Lyso-PS nanoparticles by intestinal M cells for effective antigen presentation, and subsequently oral tolerance induction. Collectively, the distinct and favorable biophysical characteristics allow Lyso-PS nanoparticles to induce effective tolerance when given via oral route.

2.2. Oral Tolerance towards rhGAA in Pompe Disease Mouse Model

The oral tolerance induced by single-chain Lyso-PS nanoparticles, but not double-chain PS, was also observed with another therapeutic protein, rhGAA, which is the first-line treatment for Pompe disease, a lysosomal storage disorder.[18] The incidence of immunogenicity in patients receiving rhGAA is 89–100% [45, 46]. Similar to FVIII, continuous oral administration with Lyso-PS-rhGAA in GAA-knockout mice resulted in significant suppression of anti-rhGAA antibodies in all animals.[18] However, the administration of four weekly SC injections of OVA, an unrelated antigen, to these animals still led to a strong development of anti-OVA IgG antibodies. This specifies that the immunoregulatory effect of Lyso-PS is specific to the pre-exposure proteins, as the immune system can still respond normally to foreign antigens or threats.

2.3. Oral Tolerance towards Ovalbumin

In addition to FVIII and rhGAA, the tolerogenic potential of Lyso-PS nanoparticles was also established with OVA, a protein found in egg white and involved in egg allergy pathogenesis. Egg white allergy affects approximately 0.9% of the children population and 0.4% of infants less than a year old in the United States.[47] Consumption of egg white in allergic children can lead to severe adverse reactions, and in some cases, fatal anaphylactic responses. Consistent with the results obtained with FVIII and rhGAA, Lyso-PS nanoparticles also led to a substantial reduction of total anti-OVA IgG antibodies in Swiss Webster mice.[18]

2.4. Oral Tolerance towards Type II Collagen

Rheumatoid arthritis (RA) is a chronic inflammatory disease that can be caused by autoimmunity against type II collagen (CII), a major protein found in cartilage. Persisting autoantibodies against CII have been detected in the sera and synovial fluid of RA patients.[48–50] More importantly, autoantibodies are present in up to 70% of early RA cases.[51] Based on positive results of previous clinical trials, where oral consumption of CII reduced inflammatory responses and ameliorated RA,[52–54] a proof-of-concept study was performed to compare the immune responses following oral treatment of CII and Lyso-PS-CII in Swiss Webster mice. The result showed that mice receiving oral treatment with Lyso-PS-CII developed significantly lower anti-CII titer levels than those treated with free CII (Fig. 2A, unpublished data). Given the superior tolerance induction potential than oral administration with just CII alone, Lyso-PS nanoparticles stand as a promising immunotherapy to treat and potentially reverse RA, as well as other autoimmune disorders.

Figure 2: Lyso-PS nanoparticles reduce immunogenicity of multiple antigens.

Swiss Webster mice were continuously pre-exposed to either 1 μg antigens alone or antigens associated with Lyso-PS nanoparticles via oral gavage for 9 weeks. Starting at week 6, mice also received four weekly subcutaneous rechallenge injections with 1 μg free antigens 24 h post oral treatment. After a 2-week wash-out period, blood was collected for titer analysis. Titer levels against (A) type II collagen and (B) AAV8 immunogenic peptide were presented as mean ± SEM. Statistical significance was denoted by P < 0.05 (*) or P < 0.005 (**) by an unpaired t-test.

2.5. Oral Tolerance toward Adeno-associated Virus Serotype 8 (AAV8) Used in Gene Therapy

The progression of gene therapy over the years with substantial achievements offers a huge potential for the treatment and prevention of a wide range of diseases. Although different types of viral vectors have been investigated for the delivery of genetic materials to target cells, adeno-associated viral vectors (AAV) remain one of the most popular delivery platforms due to their favorable biological and biophysical characteristics.[55] Among the thirteen AAV serotypes, AAV serotype 8 (AAV8) is gaining more interest over the others due to the low percentage of pre-existing antibodies against the capsid and the ability to target different muscle types.[56, 57] However, challenges arise in terms of immune activation against both the vector capsids and transgene products. Humoral and cellular immune responses against AAV8 protein capsid and therapeutic gene products have been observed in clinical trials, leading to the clearance of circulating capsids and the attack against transduced cells by antigen-specific CD8⁺ T cells, hence causing immune-mediated toxicities.[58–60] Efforts in the mapping of antigenic epitope on AAV8 capsid surface by Gurda et al. suggested that the AAV variable region containing amino acids 586 to 591 (AAV8_586–591) is the major relevant immunogenic epitope of AAV8 vector.[56] In a proof-of-concept study, in which Swiss Webster mice were continuously pre-exposed to Lyso-PS nanoparticles encapsulating the immunogenic peptide AAV8_586–591, a significant reduction of total immunoglobulin against this specific peptide was observed even with multiple re-administrations of AAV8_586–591 (Fig. 2B, unpublished data). This observation established the feasibility of further investigations using Lyso-PS nanoparticles to improve treatment outcomes of gene therapy. Studies in mice, in which the whole AAV8 vector is associated with Lyso-PS nanoparticles and administered orally, are in progress to validate the clinical applicability of this immunotherapy platform.

Proposed Mechanism of Lyso-PS-mediated Oral Tolerance

The proposed mechanism of oral tolerance induced by Lyso-PS nanoparticles is still under extensive investigation and our speculations are portrayed in Figure 3. Following oral administration, Lyso-PS nanoparticles distribute throughout the GI tract and gain entry into the LP (1) through intact enterocytes and (2) by M cells. Due to their small size, Lyso-PS nanoparticles carrying antigens can travel across the enterocytes via passive phagocytosis.[61] M cells on Peyer’s patches also act as a portal for entry of Lyso-PS nanoparticles across the mucosal barrier to LP.[17] In LP, antigens associated with Lyso-PS nanoparticles can be processed by the tolerogenic CD103⁺ DCs expressing TIM-4 or long-lived TIM-4⁺CX3CR1^hi macrophages, which then transfer antigen to the neighbor migratory CD103⁺ DCs in a connexin 43-dependent manner.[22] In the Peyer’s patches, Lyso-PS nanoparticles exiting M cells can be recognized and processed by different mononuclear phagocytes residing within the M cell pocket, including CD103⁺ DCs, CX3CR1^hi macrophages, and TIM-4⁺CX3CR1^hi macrophages. A likely alternative route of antigen sampling is via the extended projections of CX3CR1^hi macrophages along the tight junctions to the luminal side.[22] Once phagocytosed by APCs through TIM-4 and integrin signaling, Lyso-PS can engage the intracellular AhR pathway for the subsequent induction of T_regs. Following antigen presentation, tolerogenic CD103⁺ DCs upregulate the CCR7 expression and migrate via afferent lymphatics to the MLN and promote the differentiation of naïve T cells into antigen-specific T_regs, including TGF-β-producing LAP⁺ T_regs, also called TH3, and IL-10-producing type 1 regulatory T cells (Tr1).[18] These T_regs then leave the MLN via efferent lymphatics and enter the blood circulation to migrate back to the laminar propria upon the upregulation of gut-homing markers such as α4β7 integrin and CCR9.[62] A portion of these T_regs without the gut homing markers will enter the periphery for induction of systemic tolerance. The expansion of Lyso-PS-mediated T_regs subsequently suppresses the activation, differentiation, and proliferation of effector T cells, B cells, and antibody-producing plasma cells [18] via TGF-β and IL-10 production. Additionally, Lyso-PS-mediated oral tolerance involves aryl hydrocarbon receptor (AhR) signaling to modulate T_regs generation, as the pre-treatment with a potent AhR inhibitor before the administration of Lyso-PS-FVIII reversed the antibody-lowering effect of Lyso-PS in HA mice (unpublished data).

Figure 3: Proposed mechanism of Lyso-PS-mediated oral tolerance.

Lyso-PS nanoparticles gain entry into the laminar propria (1) through intact enterocytes and (2) by M cells. In the laminar propria, antigens associated with Lyso-PS nanoparticles can be processed directly by TIM4⁺CD103⁺ DCs or long-lived TIM-4⁺CX3CR1^hi macrophages, which are then transferred to the neighbor migratory CD103⁺ DCs in a connexin 43-dependent manner. In the Peyer’s patches, Lyso-PS nanoparticles exiting M cells are processed by CD103⁺ DCs, CX3CR1^hi macrophages, and TIM-4⁺ CX3CR1^hi macrophages. The third route of antigen presentation likely occurs is via the extended dendritic projections of CX3CR1^hi macrophages across the mucosal epithelium. Following antigen presentation, tolerogenic CD103⁺ DCs migrate to the mesenteric lymph nodes and promote the expansion of antigen-specific T_regs. These T_regs then either migrate back to the laminar propria for local tolerance induction or enter the periphery to induce systemic tolerance. T_regs reduce anti-drug antibody formation by suppressing the effector T cells, B cells, and antibody-producing plasma cells via the production of anti-inflammatory cytokines.

Conclusions

With unique biophysical characteristics, Lyso-PS nanoparticles display distinctive tolerogenic properties to induce effective oral tolerance via the generation of antigen-specific T_regs. Based on the rationale that pre-exposing a protein in the context of PS-based nanoparticles induces tolerance and blocks the patients’ immune response towards that protein prior to the initiation of the therapy, this approach stands as an encouraging supplement replacement therapy during the initial exposures to prevent immunogenicity and associated clinical complications. The ability of Lyso-PS nanoparticles to induce effective oral tolerance to a broad range of antigens establishes the feasibility of a user-friendly multifunctional immunotherapy platform that can be extended and applied to a wide range of clinical applications. This includes solving the immunogenicity issues of life-saving therapeutic proteins, prevention of innate and cellular immune responses against vector capsids and transgene products before the start of gene therapy, or reduction of immune reactivity against autoimmune antigens and allergens to delay disease onset and lessen disease severity.

Abbreviations

AAV8

Adeno-associated virus serotype 8

AhR

aryl hydrocarbon receptor

APCs

antigen-presenting cells

CII

type II collagen

DCs

dendritic cells

FVIII

factor VIII

GALT

gut-associated lymphoid tissue

GI

gastrointestinal

HA

hemophilia A

IV

intravenous

LP

lamina propria

Lyso-PS

lysophosphatidylserine

Lyso-PS-FVIII

FVIII associated with Lyso-PS nanoparticles

MLNs

mesenteric lymph nodes

MOG_35–55

myelin oligodendrocyte glycoprotein

OVA

ovalbumin

PS

phosphatidylserine

PS-FVIII

FVIII associated with PS nanoparticles

RA

rheumatoid arthritis

rhGAA

recombinant human acid alpha-glucosidase

SC

subcutaneous

TIM

transmembrane immunoglobulin and mucin domain

TAM

trio receptors Axl, Tyro3, and Mer

T_regs

regulatory T cells

Tr1

type 1 regulatory T cells