Retinal transplant immunology and advancements

Summary

Several gaps and barriers remain for transplanting stem cells into the eye to treat ocular disease, especially diseases of the retina. While the eye has historically been considered immune privileged, recent thinking has identified the immune system as both a barrier and an opportunity for eye stem cell transplantation. Recent approaches leveraging scaffolds or cloaking have been considered in other tissues beyond immune suppression. This perspective paper outlines approaches for transplantation and proposes opportunities to overcome barriers of the immune system in stem cell transplantation in the eye.

This perspective summarizes immunological considerations when transplanting cells into the eye, focusing on the retina. While the eye is considered “immune-privileged,” it can vary by compartment, transplantation techniques, and cell sources involved. In this article, Perez and colleagues identify needs and opportunities to successfully transplant cells in the retina accounting for the role of the immune system.

Introduction

Different parts of the eye are subject to injury or degeneration resulting in visual dysfunction. Unlike some animals, humans lack a substantial neuro-regenerative response to repair injured or degenerating retinal tissue (Goldberg and Barres, 2000; Ohlund et al., 2017). Despite significant advancements in our understanding of the pathology of retinal degenerative diseases, treatment options aimed at halting or reversing damage to the ocular tissues remain limited. The potential transplantation of stem cell-derived tissues aimed at alleviating the progression of these disorders has generated interest. However, sizable challenges remain to translate research findings into clinically effective therapeutic strategies. Among these challenges is the development of safe and reproducible methods for transplanting cells into the retina with reliable outcomes. Ongoing efforts to enhance our understanding of these methodologies may ultimately allow us to harness the potential of retinal transplantation as a dependable treatment for the millions of people affected by debilitating retinal degenerative conditions.

Collaborative efforts aimed at advancing our understanding of neural regeneration in retinal tissue, including the use of transplantation, are already underway. To this end, the National Eye Institute (NEI) established the Audacious Goals Initiative (AGI) in 2012 to stimulate research in regenerating neurons and neural connections in the retina with a focus on photoreceptors, retinal pigment epithelium (RPE), and retinal ganglion cells (RGCs). Transplant immunology involves two of the seven areas of emphasis in the NEI Strategic Plan (Immunology and Eye Health and Regenerative Medicine) and was the focus of a workshop conducted in July of 2022 (see Table 1). These ongoing efforts reflect the mounting interest in improving our understanding of the immunological aspects of retinal transplant methodologies driving development of potential therapeutic strategies against retinal degeneration.

Table 1.

Major discussion points and future perspectives derived from the panel of the National Institutes of Health’s Audacious Goals Initiative workshop on transplant immunology (July 2022)

Major Discussion points	Discussion highlights	Future perspectives
Ocular transplant immunology	The body’s immune response to transplanted tissue is non-binary and is influenced by local recipient site factors, graft formulation, and donor-to-host immunogenic interactions	Methods to achieve the target balance between pro- and anti-inflammatory responses that determine immune tolerance and rejection
Role of tissue microenvironment	The immune and cellular components at the local transplantation site play different roles that can either promote or control rejection	Animal models that better simulate the complex immune environment of humans and allow for immune tracking
Ocular immune response to allo-antigens and regenerative immunology	Engineering an immune-privileged recipient site through immunomodulation is costly but could be an option to control the immune response and rejection	The development of more affordable and accessible transplantation and immunomodulatory techniques to control inflammation and reduce rejection
Impact of the type of transplantation on the immune response	The choice of transplanted material could be altered through different formulations and biomaterials. The choice of source heavily impacts the transplant success	Better understanding of graft-related factors that influence successful integration and engineering options that improve success

This review summarizes current research developments focusing on articulating barriers and strategies in transplant immunology that may advance retinal and total human eye transplantation.

Corneal transplant immunology and its relevance in retinal tissue transplantation

A variety of concepts regarding our understanding of the immunology of tissue transplantation in the retinal tissues of the eye can be extrapolated from other forms of tissue transplantation, including those in other parts of the eye. In particular, there is a need to prevent tissue damage associated with an excessive inflammatory immune response. In healthy eyes, this is accomplished by physical anatomical barriers along with molecular means, where an immunosuppressive ocular environment composed of specific cells and soluble macromolecules can attenuate the immune response.

Covering the ocular surface, the cornea has been classically described to possess a distinctive “immune-privileged” environment due to a variety of anatomic and physiologic factors which influence the host’s response to transplantation of this tissue. As a result of this “privilege” and consequential reduced immune rejection, corneal transplantation is one of the most widely performed procedures worldwide (Niederkorn, 2003; Mousa et al., 2021; Moffatt et al., 2005). One of the main factors governing such a unique immune milieu is the anatomical absence of vascularization which typically inhibits immune cell infiltration. Infiltration of vascular and lymphatic tissue into the cornea is a major factor for increased risk of rejection allowing sensitization of the immune system (Foulsham et al., 2018). Studies that have used allo-transplants in mouse models have demonstrated sensitization and early corneal graft rejection occurring as early as postoperative day (POD) 2 (Foulsham et al., 2018). This mimics results seen in patients whereby the presence of deep stromal vessels in two or more quadrants deems the penetrating keratoplasty as “high-risk” (Fink et al., 1994). Not only that, but studies suggest that pre-operative reduction of neovascularization can decrease the risk of rejection compared to “normal-risk” corneal transplants (Hos et al., 2019).

Aside from anatomic factors, the presence of macromolecules including FasL and PDL-1 can also reduce inflammation and play a part in controlling the immune environment of the cornea. Because of such factors and the attenuation of allo-sensitization to donor tissue antigens, human leukocyte antigen (HLA) matching has shown no benefit in corneal transplantation and is not necessary as in other tissue/organ transplantations (Fink et al., 1994). That said, it is important to highlight that, even in non-diseased eyes, immune cells do reside in the cornea and can instigate a pro-inflammatory response to antigens deeming the term “immune privilege” to be not fully accurate. Therefore, immune-mediated graft rejection, namely by a Th1 response, still occurs and remains the most common cause of corneal graft failure (Foulsham et al., 2017).

Nevertheless, considering that the aforementioned factors that reduce sensitization to donor allo-antigens in the cornea are not as evident in the posterior segment of the eye, it is reasonable to posit that the vascularization and sensitization to allo-antigens will be an obstacle to the success of transplantation of retinal tissue and cells. Understanding the unique factors that limit the immune responses in corneal transplantation and the absence of such factors in the posterior segment can guide strategies to optimize retinal tissue transplantation outcomes by reducing immune rejection.

Considerations in retinal transplantation

Global population growth and aging contribute to the high prevalence, and the number of people suffering visual impairment is projected to increase. The etiology of vision loss due to retinal disease or trauma is variable, and damage to the retina and other ocular tissues often leads to vision loss. How the distinct retinal pathologies might influence the immune response after transplantation requires consideration when devising effective transplantation strategies to restore the retina. For example, diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) result in damage to the photoreceptors and the RPE, a monolayer of pigmented cells critical for maintaining the light-sensitive photoreceptors essential for vision (Keam, 2022). AMD, diabetic retinopathy, and glaucoma all have retinal ischemia as a pathomechanism.

The progression of these diseases can greatly affect the immune system. For example, in RP mutations photoreceptor function is disrupted with loss of rod photoreceptors in the periphery followed by secondary cone loss. Although the RPE is largely intact, regions of photoreceptor degeneration result in chronic inflammation releasing cytokines that attract resident microglia and infiltrating myeloid cells (Newton and Megaw, 2020). The speed of vision loss associated with RP can vary with rapid onset potentially limiting transplantation success due to the degree of glial scarring and outer limiting membrane (OLM) disruption (Newton and Megaw, 2020). Additionally, complete photoreceptor loss is also accompanied by extensive structural remodeling of the retina (Jones et al., 2016) suggesting early therapeutic intervention may be required (Wu et al., 2023).

In contrast, diseases of the RPE, such as AMD, are multi-factorial with genetic, behavioral, and environmental risk factors. Early features of AMD include drusen accumulation between the RPE and Bruch’s membrane consisting of lipids, proteins, and pro-inflammatory components that can cause inflammasome activation (Ambati et al., 2013). Dysregulation of the complement pathway has also been implicated in AMD pathogenesis; as the disease progresses, there is massive disruption of the RPE monolayer resulting in breakdown of the blood-retina barrier (BRB) which can lead to geographic atrophy (GA) or choroidal neovascularization (CNV), each with different immunological contributions. In GA, there is RPE atrophy and autofluorescent lipofuscin accumulation coinciding with photoreceptor loss (Wong et al., 2022); however, there is no significant immune cell infiltration as seen in CNV. Thus, transplantation efforts must consider the stage of the disease and whether circulating immune cells have access to the degenerating retina, and if their presence enhances or hinders repair.

Glaucoma encompasses several optic neuropathies that are associated with elevated intraocular pressure (IOP). At the optic nerve head, chronic ischemia eventually damages the optic nerve and causes RGC death. Therefore, glaucoma can be considered as an ischemic optic neuropathy (ION), classified as either anterior (AION) or posterior (PION) and further separated into arteritic (AAION, APION), nonarteritic (NAION, NPION), and perioperative (PeAION, PePION) types (Bernstein and Miller, 2015). Ischemia in the eye can be acute or chronic, resulting in oxidative stress-induced damage, inflammation, and activation of an immune response. Animal models of both acute retinal ischemia followed by reperfusion using transient elevation of IOP and chronic ischemia by artificially elevating IOP are well established (Adler, 1996; Anderson and Hendrickson, 1974; Hayreh, 1969; Morrison et al., 1997; Quigley and Addicks, 1980). These studies have shown that ischemia produces changes throughout the retina, impacting the viability of various cell types including photoreceptors, amacrine cells, and RGCs (Chan et al., 2012; Lafuente et al., 2002; Levin, 1999; Nickells, 1996; Palmhof et al., 2019; Tezel and Wax, 2000). Since ischemic damage to donor organs is unavoidable and associated with all current allotransplantation, regenerative strategies that are accompanied by ischemia, such as whole-eye transplantation, should consider attenuation of the inflammatory load and immuno-response after transplantation. Immune response differs in retinal degenerative diseases depending on whether the BRB is damaged. In diseases with breached BRB, accumulation of fluid in the retinal parenchyma results in edema or a “wet” retina. Neovascular AMD, uveoretinitis, diabetic macular edema (DME), and macular edema secondary to retinal vein occlusions (RVOs) are all “wet” retinal diseases. When the BRB is dysfunctional, circulating immune cells can infiltrate and plasma proteins accumulate in the parenchyma, exacerbating an inflammatory state that leads to retinal degeneration (Xu and Chen, 2022). In “dry” retinal degenerative disease the BRB is functionally intact and still provides a physical barrier between the blood and the RPE. Thus, the retina remains “dry,” and microglia and the retinal complement system remain quiescent. Patients with dry retinal degeneration present as a slow loss of visual function and retinal thinning. Dry retina diseases include RP, glaucomatous retinopathy, and GA type of AMD (Xu and Chen, 2022).

As the retina ages, there is an increased frequency of a chronic inflammatory state, which disrupts normal immunoregulatory activity of endothelial and glial cells. (Chen et al., 2019). Mononuclear phagocytes-derived interleukin (IL)-1β has been shown to cause photoreceptor cell death during experimental subretinal inflammation and in retinal explants (Charles-Messance et al., 2020). Additionally, Müller cells from aging eyes contain high levels of oxidized lipid (4-hydroxy-2-nonenal), indicative of the pathological state of the aged retina. (Li et al., 2022).

In the back of the eye, promising approaches using allogeneic human induced pluripotent stem cell (hiPSC)-derived RPE transplants have led the way for advances toward clinical restoration of retina-associated vision loss. One of these interventions involves formulation of transplanted RPEs, which can be delivered as a cell suspension or a graft (Binder et al., 2002). Cell suspension RPE transplantation is generally successful, requiring smaller retinotomies but maintains the risk of reflux of transplanted cells into the vitreous cavity. Changes to Bruch’s membrane with age or because of the fact that disease can perturb cell attachment makes it less likely for cell suspensions to form an effective monolayer with the correct orientation and polarization to match the native RPE. On the other hand, graft RPE transplantation allows pre-orientation and establishment of a fixed treatment area; however, placement of such grafts requires specialized surgical tools and a more complicated surgical procedure (Osakada et al., 2009).

Overall, survival of RPE cell suspension transplants is lower than that of scaffolded RPE transplants. RPE cells in suspension lack normal epithelial cell-cell contacts and have rounded rather than hexagonal morphology, an indication of being more distressed. When RPE cells lose cell-cell contact, they are vulnerable to dedifferentiation and epithelial-mesenchymal transition (EMT) (Zou et al., 2020). Thus, supporting factors from the extracellular matrix (ECM) and adhesion molecules likely contribute to the improved cell survival on scaffolds (Lukomska et al., 2021). The consequence of poor survival of cell suspension transplantation is that higher dosing may be required, which can result in safety issues. A comparison of iRPE delivered by cell suspension versus iRPE patch on poly (lactide-co-glycolide) scaffolds demonstrated that iRPE cell suspension failed to integrate with host tissue in an immunocompromised rat, whereas at 10 weeks post-surgery the iRPE patch formed a contiguous monolayer that was negative for the Ki67 proliferation marker (Sharma et al., 2019). Scaffold composition can influence cell-to-cell and cell-to-scaffold interactions as well as the immune response. Nonbiodegradable scaffolds prevent implanted RPE from forming a contiguous border with the host RPE (Diniz et al., 2013) whereas the byproducts of synthetic biodegradable materials may cause inflammation or alter the microenvironment requiring careful selection and testing (Stratton et al., 2016). As an alternative approach to overcome such limitations, the RPE cell strip transplantation method was developed whereby the transplanted cells are generated as a monolayer without any scaffold (Kamao et al., 2014). This approach was tested in monkeys without immunosuppression and showed that, when the recipient animal was major histocompatibility complex (MHC) mismatched, the graft showed typical signs of rejection (e.g., fibrosis and edema), whereas the graft survived in the autograft recipient and showed no evidence of tumor formation. In monkeys, a method for human RPE cell sheet transplantation via a bullet-shaped polyester scaffold allowed for a stable graft with reduced gliosis 3 months post-operatively (Luo et al., 2021). Biological scaffolds derived from decellularized ECM have the potential to combine non-immune environments with native 3D bioactive structures (Zhang et al., 2022). However, to date decellularized retinas currently lack the mechanical strength to maintain structure and conformation upon repopulation (Maqueda et al., 2021) limiting their therapeutic potential but provide an excellent model to study how extrinsic cues in the ECM regulate cellular organization. Aside from the route of delivery of the transplant, other considerations include options that reduce or control the immune response to transplantation. As previously highlighted, HLA matching is not critical for corneal graft survival in transplantations. However, determining the role of donor-recipient HLA matching in RPE graft survival has had promising implications. In a study using a primate animal model, transplantation of HLA-unmatched hiPSC-derived RPE cells into monkeys required subsequent immunosuppression to prevent inflammation and retinal thinning, whereas HLA-matched hiPSC-derived RPEs survived after transplant beneath monkey retinas without the need for immunosuppression (Sugita et al., 2016). This result was translatable to humans in which HLA-matched allogeneic hiPSC-derived RPE transplants were successful in a five-patient clinical trial.

Using co-culture experiments, HLA-DR, an MHC class II cell surface receptor, was found to be a key determinant of RPE-transplant immune reactions by stimulating retinal antigen-specific T cells. On the other hand, postmortem histology of a patient with GA that received a subretinal implant of HLA-mismatched human embryonic stem cell-derived RPE monolayer on a parylene membrane showed survival of the RPE, expression of makers of RPE cell polarity, and no clinical evidence of inflammation even in the absence of long-term immunosuppression (Humayun et al., 2023).

This suggests controlling the immune responses during the first 60 days following allogenic implantation may improve survival by providing sufficient protection during the period when inflammatory responses are expected to peak.

In Japan, the establishment of HLA-homozygous iPSC banks has the potential to revolutionize regenerative medicine and improve the success of iPSC-based therapies. Such banks utilize iPSCs derived from individuals who are homozygous for a particular HLA allele that can cover a significant portion of the Japanese population, providing marked versatility in care of different patients (Nakatsuji et al., 2008). Learning from other solid organ transplantations (SOTs), the most important HLA molecules to consider for matching include HLA-A, HLA-B, and HLA-DR (Zachary and Leffell, 2016). In Japan 80% of patients are expected to be able to find at least one ES cell line that has only a single HLA mismatch from a bank of 170 embryonic stem cell lines derived from randomly donated embryos (Nakajima et al., 2007). A similar study was performed previously in the UK from cadaveric organ donors consisting of 77.0% White, 12.0% Asian, and 7.0% African donors. In this study 150 donors provided a full match for 18.5% of patients and a beneficial match (one HLA-A or one HLA-B mismatch only) for 37.9%. In the United States, a study that modeled the number of iPSC lines required to establish a haplobank for the ethnically diverse state of California found that 50% of the simulated population would be covered with 207 haplolines representing the top 60 most frequent haplotypes from each ancestry group (Pappas et al., 2015). Researchers in the gene therapy space and cell transplantation space would benefit from interaction to overcome immune system barriers and improve the effectiveness of retinal therapies. Recent work by some groups has shown that both the innate and adaptive immune systems play a role in a response to adeno-associated virus (AAV) gene therapy approaches in primates. Some similarities to overcome immune issues from either gene therapy or cell therapies are immunosuppression methods such as rapamycin and oral prednisone, a temporary shielding of the injection site from the immune system, or targeting the antigenicity of AAVs or donor cells. Clearly the immune system is still a barrier to more effective AAV and cellular therapies.

Role of tissue microenvironment in axonal repair and regeneration

Different eye compartments (e.g., outer versus inner retina, retina versus optic nerve) consisting of different microenvironments likely respond to transplants differently, especially in response to transplantation-related trauma. For compartments less tolerant of transplants, strategies that can modify the microenvironment may improve transplant acceptance rate. Alternatively, before transplantation, donor cells could be modified to reduce disruption to the transplant site. In such an approach, patient-related factors such as aging, cellular senescence, trauma, inflammation due to disease, and rewiring of retinal circuitry because of disease also need to be taken into consideration.

Another factor to consider is the type of resident cells at the transplant site and their cell-to-cell interactions. Resident immune cells interact with transplanted cells via multiple mechanisms, including secretion of various factors, formation of cell-cell contacts, and exchange of cellular material. Different cell types in the tissue microenvironment affect axonal repair and regeneration (Rickman et al., 2008). Some inferences can be extrapolated from studies on other organs to identify potential therapeutic targets. For example, after spinal cord injury, neutrophils promote a scarring response to wall off the lesion and prevent spread of damage, and macrophages may play an important role in repair and regeneration (Fan et al., 2018). Due to the heterogeneity of glial cells (which include microglia, astrocytes, and Müller glia), the extent of their involvement in axonal repair and regeneration is not well understood. Further studies of these mechanisms will help determine which, if any, are important for axonal repair and regeneration. A deeper spatiotemporal characterization will provide insights into glial cells’ roles in these processes.

An additional area of interest is the impact of immune regulation on the microenvironment and its positive or negative effects on transplant success. Immediately after ocular transplantation, general immunosuppression may be required for initial transplant survival. However, as previously highlighted, systemic immunosuppression can negatively affect integration and regeneration of transplanted cells. For example, induction of sterile inflammation in an optic nerve crush murine model triggers optic nerve axon regeneration (Wong and Benowitz, 2022). Therefore, after the initial period following transplantation, immunomodulation should shift the immune system toward pro-regenerative functions over immune rejection functions. Research has been carried out to identify factors involved in promoting innate immune system-driven pro-regeneration. Researchers have identified a fungal cell extract compound, β-1,3-glucan, that binds Dectin-1 on retinal microglia and resident dendritic cells. This binding triggers the release of chemokines and vasoactive substances that recruit myeloid cells to the vitreous body promoting axon regeneration (Baldwin et al., 2015). Similar immature myeloid cells derived from bone marrow precursors and delivered to the vitreous body via extracellular vesicles trigger a similar axonal regenerative response. However, results from other studies indicate that innate immune system functions and increased microglia may suppress regeneration (Todd et al., 2020). These conflicting results may suggest that only specific subpopulations of microglia and other immune cells can promote axon regeneration.

Ocular immune response to allo-antigens and regenerative immunology

Differences exist between the various types of transplantation and the tissues that they replace (Yu et al., 2016). However, the molecular events associated with allograft rejection that begin with ischemia-reperfusion injury (IRI) stimulation of the innate immune system and activation of the adaptive immune system, allo-recognition, and graft tissue destruction are all cellular-mediated mechanisms. Identifying the cells, their spatiotemporal details, and functional roles during the alloresponse to transplantation is the foundation for rational immunosuppressive strategies and targeted therapeutics capable of inducing long-lasting, allograft-specific tolerance. Strategies for modulating the ocular immune response can potentially improve stem cell transplantation outcomes. The field of ocular transplantation can gain relevant insights from related fields of study including cancer immunotherapy, vascularized composite allotransplantation (VCA), SOT and other cellular transplantation, and aging (Abud et al., 2017). For example, immunosuppression regimens in SOT are well established and have been shown to improve long-term outcomes of allografts. Pharmacological immunosuppression is the current gold standard for SOT and VCA patients, with most surgical centers employing a regimen of induction therapy (e.g., anti-thymoglobulin; alemtuzumab) followed by maintenance immunosuppression consisting of tacrolimus/FK506, mycophenolate mofetil (MMF), and prednisone. FK506 (tacrolimus) is a widely used Food and Drug Administration (FDA)-approved immunosuppressive drug in SOT and VCA to improve graft survival and reduce rejection (Schnider et al., 2013). Use of systemic and topical FK506 to treat ocular disorders has demonstrated efficacy in corneal graft rejection, inflammatory conjunctival and corneal diseases, uveitis, and graft-versus-host disease (Bertelmann and Pleyer, 2004; Sengoku et al., 2003; Sloper et al., 1999; Zhai et al., 2011). However, adoption of high-dose systemic FK506 for maintaining immunosuppression in organ transplantation is complicated by end-organ adverse effects including rejection, increased risk of chronic renal disease, diabetes, infection, osteonecrosis, and malignancy (Davaus Gasparetto et al., 2010; Dehghani et al., 2010; Diaz-Siso et al., 2016; Gallagher et al., 2010; Bäckman, 2004; Leroy et al., 2010; Ojo et al., 2003; Oto et al., 2010; Tricot et al., 2005). Strategies to reduce systemic exposure and limit end-organ damage include 1) local delivery of immunosuppressive agents to targeted tissues and 2) immunosuppressive drug monotherapy (Gama et al., 2020; Hasan et al., 2022; Rosenstiel et al., 2003; Safi et al., 2021; Xiao et al., 2024).

Topical corticosteroids are the most common medication prescribed to prevent immunologic rejection after keratoplasty, but there are important differences in the postoperative management protocols (Kharod-Dholakia et al., 2015). Although weaker steroids may have better safety profiles, some patients will require the strongest available drug to control their inflammation (Aydin et al., 2004; Doolabh and Mackinnon, 1999; Fansa et al., 1999; Jost et al., 2000). The most widely prescribed topical corticosteroid in the world is prednisolone acetate 1% (Sinha et al., 2010). Despite their efficacy, the ocular side effects of long-term topical steroid use are well known and include elevated IOP and cataract formation. To address these concerns, alternative immunosuppressants and approaches have been considered and investigated (Cozzi et al., 2017).

One immunomodulatory strategy involves engineering the immune-privileged site at the locus of transplantation. Privileged site engineering would require addressing multiple factors that affect the immune response, including transforming growth factor β (TGF-β), neuropeptides, and maintaining isolation from the immune system. A proposed method for such an approach involves examination and elucidation of the regulatory T cell (T-reg) response to ocular tissue grafts. The eye naturally converts T cells into T-regs, and amplification of this conversion could be beneficial. Additional strategies for engineering an immune-privileged site could include co-transplantation with immunosuppressive stem cells or exosomes. Activation of T-regs can successfully manage graft-versus-host disease, since T-regs and regulatory B cells (B-regs) have both been shown to produce exosomes capable of suppressing inflammation in the spinal cord, brain, and retina. These anti-inflammatory exosomes also extend murine model survival after bone marrow transplantation. A similar exosome delivery mechanism may successfully modulate the immune response to ocular transplantation. However, current ex vivo T-reg production is currently complicated by cost, functionality, and durability. Instead, therapeutics can be aimed to bias the host immune system toward T-reg production in vivo. For example, at very low doses IL-2 triggers phosphorylation of STAT5 in T-regs but not in other T cell populations, enabling selective expansion and activation of T-regs to release modulatory exosomes. Alternatively, because B-regs can also induce T-regs, treatment using B-regs may result in amplified transplant tolerance compared to T-reg therapeutic strategies (Schreiber et al., 2010).

Another strategy for achieving appropriate immune modulation involves the engineering of universal donor cells. Past studies identified FasL secretion as a trigger for apoptosis. To generate a universal donor-type tissue that would defend itself against any immune cell encounter, researchers have engineered FasL-expressing cells (Li et al., 2002). However, this approach was ineffective because these cells activate an inflammatory response and attract neutrophils. A recent publication described the coupling of FasL to islet cells via streptavidin prior to transplantation, and this coupling sufficiently defended against the immune response without triggering an inflammatory reaction. However, researchers have not yet developed this particular strategy further (Yolcu et al., 2011). Rather efforts have been made to generate immune-cloaked stem cell therapies by deleting HLA expression in its entirety by removing beta-2-microglobulin (B2M) and class II transactivator (CIITA) genes which effectively suppresses all HLA-class I and HLA-class II expression, respectively (Koga et al., 2020). However, the suppression of these genes, notably B2M, prevents the surface expression of HLA-E and HLA-G, which leads to enhanced natural killer (NK) cell activity. Strategies to combat this response include forced expression of HLA-E and CD47, known NK inhibitory ligands.

Recently, CRISPR-Cas9 system-specific deletion of only HLA-A/B expression from CD43⁺ blood cells while retaining minor HLA-class I molecules (unmodified HLA-C/E/F/G) was shown to evade immune rejection from only HLA-C-matched NK cells and T cells, in in vitro and in vivo experiments (Koga et al., 2020).

Taking this one step further, Han et al. targeted for deletion of HLA-A/B/C and CIITA molecules while simultaneously expressing PD-L1, HLA-G, and CD47 to evade immunological responses (Han et al., 2019). However, some residual activation of T cells was still observed in vivo (Han et al., 2019). A challenge moving forward is achieving and evaluating hypoimmunogenicity of genetically engineered pluripotent stem cells while ensuring their safety (reducing the risk of transmittable oncogenic mutations due to loss of MHC observed in other species, e.g., Tasmanian devils) (Zhao et al., 2020). Additional banking strategies targeting common antigens in specific populations would also be an effective strategy at minimizing risk of rejection following the Japanese example of establishing homozygous HLA donor hiPSC banks.

Impact of the type of transplantation on the immune response

The transplant procedure itself carries a significant impact on the resultant immune response and allograft outcome. For example, and particularly in scaffold transplants, surgical techniques that minimally expose the ocular microenvironment to the immune system via blood and lymph vessels can reduce the risk of ocular transplant rejection. After more invasive transplantations that likely expose the microenvironment to the immune system, heavy immunosuppression is required to prevent transplant rejection. Alterations in the decisions related to the type of transplantation can carry a substantial impact on the immune response. In that regard, the maturity, delivery, and compatibility of the transplanted cells and tissue can be altered to improve outcomes.

Regarding cell maturity, embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived cells may elicit reduced antigenicity compared to mature cells (Ankrum et al., 2014). A previous study found that mouse iPSC-derived RPEs elicited a weaker immune response compared to undifferentiated mouse iPSCs when transplanted in the muscle (Zhao et al., 2015). Therefore, manufacturing high-quality controls for iPSC-derived RPEs should ensure removal of any undifferentiated cells prior to transplantation to reduce immune responses (Mandai et al., 2017). On that note, differential immune responses to various transplanted cell types need to be considered. For example, while RPE cells are anti-immunogenic and suppress some inflammation perhaps aiding in their integration, photoreceptors are more difficult to structurally integrate with the host retina and instead participate in a highly specific photoreceptor-photoreceptor interaction called cytoplasmic material transfer (Gasparini et al., 2019). Progenitor cells may also elicit a different immune response than fully differentiated cells that have exited the cell cycle.

In terms of delivery method, scaffold-assisted transplants are more invasive than cell suspension transplants, and thus the development of specialty instrumentation for implanting scaffolds can reduce surgically induced trauma and the risk of complications (Osakada et al., 2009). For example, minor antigens drive the immune response to solid organ transplants, such as corneal transplants, whereas major antigens drive the immune response to cell suspensions (e.g., bone marrow stem cell transplants); therefore, a cell suspension corneal transplant would require immunomodulation to address the involvement of both minor and major antigens. In cases where nerve exposure is unavoidable such as VCA transplantation of the eye, scaffolds present a powerful delivery method. In particular biomaterials can be engineered for both tissue engineering and targeted delivery of beneficial trophic compounds (Lin et al., 2019; Stankus et al., 2004; Su et al., 2023; Xu et al., 2016).

When considering serologic compatibility for transplantation, xenograft models elicit a much stronger immune response than allograft models (Wang and Yang, 2012). Additionally, results from xenograft research may not be fully translatable to allografts because with xenografts serum complement activation plays a prominent role in acute rejection and complement biomarkers are not routinely screened in transplant recipients. As such, the ocular transplant field may benefit from prioritizing research and development of allograft models. One existing model for investigating the immunological response to whole-eye allotransplantation is an orthotopic model for vascularized eye transplantation in the rat (Bourne et al., 2017; Davidson et al., 2016). This high-throughput, small-animal model represents an unprecedented platform for basic and translational research providing insight into ocular transplant and regenerative immunology (Table 1).

New techniques and technologies are being developed to advance retinal transplantation, but their successful clinical implementation will ultimately depend on technical feasibility and overcoming logistical challenges. The ocular transplant field needs to develop affordable, and therefore more practical, ocular transplant options. The chemistry, manufacturing, and management of each personalized product would require extensive quality control and assurance. Additionally, new ocular transplantation techniques need to be validated with autologous proof-of-concept studies before progressing to allogeneic transplantation.

Conclusions

Discussion on the needs and opportunities in transplant immunology of the eye identified current barriers including the need for better animal models, improved surgical techniques, and a basic understanding of the ocular immune response to transplantation (e.g., immune homeostasis). Our understanding of these microenvironment interactions could be accelerated by using animal models that allow in-depth investigation. Particularly, this will require preclinical animal models that better reflect the human immune environment. Such models must extend beyond laboratory testing in mice which have a “too clean” microbiome and low levels of activated memory T cells due to isolated rearing (Willyard, 2018). Humanized mouse models such as human immune system mice (Flahou et al., 2021) might be a more tractable model that recapitulates what might happen in a human after transplantation. Alternatively, the porcine eye better emulates the human eye, and the morphology of photoreceptors is very similar; however, such models are often inaccessible to academic researchers. Potential methods to address these restrictions are transpiring. For example, The National Swine Resource and Research Center (https://nsrrc.missouri.edu/) is attempting to establish a visual function testing facility that would enable researchers to perform porcine experiments without maintaining pigs and related resources at their home institutions (Walters et al., 2012). Easier accessibility to such models would promote more clinically relevant investigations that can offer improved translatability. Opportunities identified were the use of T-regs and B-regs, exosomes, and extracellular vesicles as both regulators of the immune system and delivery methods for other immune modulators. Alternative approaches for improving our understanding of immune microenvironment interactions are through tracking immune responses and transplant survival. In scaffold-assisted transplants, the area of experimental treatment is more clearly defined than that of a cell suspension transplant, enabling easier monitoring of viability and integration. Considering that methods such as fluorescent labeling are not viable options in the clinical setting, the establishment of alternative methodologies for immune tracking would further our understanding of the outcomes in these procedures. A potential approach for immune tracking would be to utilize postmortem donated eyes with participant consent. Through these donations, clinicians and researchers have the rare opportunity to perform histology to assess transplant survival and infer functionality, but this approach does not enable transplant survival monitoring over time.

Other immunomodulation strategies were considered, including “cloaking” or scaffolds that encourage incorporation of donor tissues (see Figure 1). Collaboration between immunologists and stem cell biologists will be critical to solving these issues related to transplant immunology.

Figure 1.

Strategic approaches to immune modulation

(A) Tacrolimus (FK506), a common immunosuppressant drug, prevents tissue rejection by inhibiting T cell activation. Tacrolimus inhibits the nuclear factor of activated T cells (NFAT) pathway resulting in suppression of IL-2 gene transcription, an autocrine T cell growth factor pivotal for T cell activation. APC, antigen-presenting cell; MHC II, major histocompatibility complex (MHC) class II; IL-2. interleukin-2.

(B) Patients that are human leukocyte antigen (HLA) matched with potential donor iPSC lines can be transplanted with allogenic stem cell-derived tissue (e.g., iPSC-RPE transplant). HLA molecules are the human version of MHC, cell surface proteins that are involved in antigen presentation and recognition by the recipient’s immune system.

(C) Cloaking is a technique to engineer universal cells that can evade the immune system. Overexpression of several transgenes such as PD-L1, FASL, CD47, and CD200 can modulate immune cell activity masking the presence of donor cells and tissue.

(D) Scaffolds provide mechanical support during transplantation but can also be functionalized with “immune-calming” modulatory properties. Scaffold strategies for modulating the immune microenvironment include altering its physical properties, chemical properties, degradation rate, and oxygen concentration or embedding it with cytokines or anti-inflammatory drugs.

(E) Exosomes, nanosized extracellular vesicles, can be utilized to regulate the immune response. T-regs and B-regs can be coaxed to release exosomes that are anti-inflammatory. These exosomes can contain polypeptides, peptides, RNA, or DNA promoting immunologic tolerance.

While the eye is a unique, accessible, and “immune-privileged” tissue, there are still many immunological discoveries to be made to achieve active integration of neural stem cell transplants in the eye providing hope for people with degenerative eye diseases.