Emerging local immunomodulatory strategies to circumvent systemic immunosuppression in cell transplantation

ABSTRACT

Introduction

Cell transplantation is a promising curative therapeutic strategy whereby impaired organ function can be restored without the need for whole-organ transplantation. A key challenge in allotransplantation is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with serious adverse effects such as increased risk of opportunistic infections and the development of neoplasms. This challenge underscores the urgent need for novel strategies to prevent graft rejection while abrogating toxicity-associated adverse events.

Areas covered

We review recent advances in immunoengineering strategies for localized immunomodulation that aim to support allograft function and provide immune tolerance in a safe and effective manner.

Expert opinion

Immunoengineering strategies are tailored approaches for achieving immunomodulation of the transplant microenvironment. Biomaterials can be adapted for localized and controlled release of immunomodulatory agents, decreasing the effective dose threshold and frequency of administration. The future of transplant rejection management lies in the shift from systemic to local immunomodulation with suppression of effector and activation of regulatory T cells, to promote immune tolerance.

KEYWORDS:

• Local immunosuppression
• cell therapy
• immunomodulation
• long-acting drug delivery

1. Introduction

Advances in cell transplantation promise to restore lost cellular function and reverse diseases that are currently incurable. Advantages of cellular over whole organ transplantation include less tissue mass replacement, minimally invasive procedures, and opportunities for graft immunoengineering. However, with the exception of hematopoietic stem cells (HSCs) [1], cell transplantation strategies have encountered limited success in the clinic.

The major hurdle for transplantation of allogeneic cells and tissues is the requirement of systemic immunosuppression to prevent rejection, which represents a double-edge sword. Specifically, systemic immunosuppression regimens can abrogate acute transplant rejection and delay immune rejection of the allograft [2], albeit at the expense of organ toxicity, increased risk of infections, and neoplasm development [3–5]. Moreover, there is a struggle to balance optimal dosing to avoid under- and over immunosuppression. Thus, a collective goal in the modern era of transplantation is to determine effective anti-rejection approaches that present high safety profiles with minimal immunosuppression-derived toxicity [2].

The standard of care in contemporary immunosuppression regimens typically involves the use of antibody therapy for induction, followed by maintenance with a combination of immunosuppressive agents targeting various rejection pathways [6]. This strategy minimizes agent-associated morbidity while maximizing overall effectiveness. Antibody induction therapy includes T cell-depleting and non-depleting agents. Depleting antibodies are typically used as they provide aggressive immunosuppression in the initial days after transplantation, preventing acute rejection [6,7]. Common T cell depleting antibodies used in solid organ transplantation are polyclonal rabbit antithymocyte globulin (rATG; Thymoglobulin) or alemtuzumab (anti-CD52 monoclonal antibody (mAb)) [8,9]. rATG is mostly used in patients with high risk for delayed graft function (longer cold ischemia time or expanded transplant indication). On the contrary, basilizimab, a non-depleting monoclonal antibody, is used for low-risk patients. Basiliximab is directed against the interleukin-2-receptor (CD25) which is only expressed on activated T cells, inhibiting T cell proliferation and differentiation without causing depletion [10,11]. In some cases, although not as effective, induction is achieved with high doses of agents typically used for maintenance [6]. Common maintenance immunosuppression agents include calcineurin inhibitors (CNI), antiproliferative/antimetabolites, glucocorticoids, mammalian target for rapamycin (mTOR) inhibitors, and T cell costimulation blockers [6,7]. The combinations and doses of maintenance immunosuppressants vary between transplant centers, although a widely adopted regimen for whole organ transplants consists of a combination of tacrolimus, mycophenolate mofetil (MMF), and corticosteroids [2]. Maintenance immunosuppression regimens are established within the first 3 months after transplantation. Reduction in maintenance therapy is not recommended, since it frequently leads to late acute rejection of the allograft. However, some patients may require a modification of the initial maintenance regimen to avoid graft loss. For instance, long-term immunosuppression with CNI may contribute to chronic graft dysfunction due to toxicity-associated adverse events [12,13].

Toxicity-associated complications are the major drawbacks of systemic immunosuppression, ultimately leading to death of patients with functioning grafts, predominantly due to cancer (29%), followed by cardiovascular diseases (23%) and opportunistic infections (12%) [14]. CNI, which are the backbone of immunosuppressive therapy, can cause renal cell toxicity and cellular senescence [15], contributing to premature decline in allograft function in kidney transplant patients and chronic renal failure among patients with nonrenal transplant [16]. Furthermore, although glucocorticoids are effective suppressors of the immune system, they induce metabolic disorders such as hyperglycemia, hyperlipidemia, and hypertension [17]. Therefore, a more tailored approach is needed to mitigate adverse events associated with standard immunosuppression.

In this context, using immunomodulatory therapies to achieve immune tolerance instead of suppression have gained significant interest. Creating an immune tolerogenic microenvironment decreases the risk of acute and chronic graft rejection after solid organ transplantation and can improve transplant survival. Negative costimulatory molecules such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), or co-inhibitory receptor B and T lymphocyte attenuator (BTLA), and CD160 are investigated for their immunomodulatory effects, as they are capable of immune response inhibition while maintaining peripheral tolerance [18]. Moreover, regulatory T cell (Treg) therapy is investigated in the clinic [19], as Tregs play a major role in inducing and maintaining immune tolerance early after transplantation [20,21].

Further, advances in biomaterials, membranes, and surface functionalization, have led to immunoengineered strategies for immunosuppression-free protection from rejection. Among these, cell encapsulation technologies can prevent rejection by creating a physical barrier between the transplanted graft and the hosts immune cells. However, poor oxygen permeability due to lack of direct transplant vascularization compromises long-term graft viability.

The aforementioned challenges yielded the premise of site-specific immunomodulation for transplantation protection [Figure 1]. This review highlights emerging localized immune modulation approaches to protect allografts, including biomaterial functionalization, local release, and co-delivery of immunomodulatory agents or cells, targeted delivery of nanoparticles, hypoimmunogenic therapeutic cells, and integration of local immunosuppression and cell transplantation within macroencapsulation systems.

Figure 1. Immunoengineering strategies to achieve local immunomodulation in cell transplantation including 1) surface functionalization of biomaterials with immunomodulatory factors, 2) immunosuppressant-loaded biomaterial platforms (i.e. scaffolds, hydrogels, nanoparticles/microparticles) for in situ deployment and release, 3) nanoparticles with surface ligands for targeted delivery of immunomodulatory agents to lymph nodes (LN), immune cells or allogeneic graft; and 4) co-transplantation of immunomodulatory cells such as mesenchymal stem/stromal cells (MSCs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) or extracellular vesicles (EVs).

2. Cell encapsulation

Cell encapsulation is an extensively researched strategy for immunosuppression-free rejection prophylaxis, specifically for islet transplantation to manage type 1 diabetes [22–26]. Cell encapsulation strategies have shown successful outcomes in clinical trials [27–29], albeit for limited duration. The overarching principle of encapsulation is to provide a physical barrier that isolates the transplanted cells from host immune attack; thus, overcoming the need for immunosuppression. Based on the scale of the encapsulation system, biomaterial-based strategies can be classified as (I) nanoencapsulation, in which cell surfaces are individually coated with a nanoscale immune isolation layer; (II) microencapsulation, where one or a few cells are embedded within a permselective scaffold or microbeads; and (III) macroencapsulation, where a large mass of cells is usually confined within an implantable device [23].

Nanoencapsulation minimizes the coating thickness and improves diffusion for oxygen, nutrients, and insulin. However, nanoscale permselectivity is minimal as it does not provide complete protection from cytokines [30]. Microencapsulation provides the advantage of a high surface-to-volume ratio, allowing efficient diffusion of nutrients and cell waste products. However, they require curative transplant volumes >100 mL, creating a major hurdle in clinical deployment as very few sites in the body can hold such large volumes in an orthotopic setting [22]. In contrast, macroencapsulation devices allow for flexibility in implantation site with potential for minimally invasive subcutaneous (SC) insertion. A potential drawback for these systems is the formation of pericapsular fibrotic overgrowth as a result of the foreign body reaction (FBR) to the device material. Fibrotic tissue formation reduces oxygen and nutrient supply to the grafted cells, which is already limited by lack of direct vascularization, creating a hypoxic environment inhospitable for long-term cell viability. Inadequate oxygen supply is a major limiting factor for clinical translation and implementation of cell encapsulation systems. Thus, enhanced oxygen delivery strategies are being actively developed and tested [31]. Some systems such as β-Air (Beta-O₂) have attempted to address encapsulation anoxia via exogenous oxygen supplementation through external ports, which has demonstrated clinical feasibility for daily refilling [32]. However, lack of direct cell apposition to blood vessels impair mass transport critical for metabolic activity, thus affecting graft viability and function [32,33].

Newly developed nanofiber technologies for islet transplantation reduced fibrotic and allogeneic responses to nanofiber-integrated cell encapsulation (NICE) devices implanted in the intraperitoneal (IP) space [34]. The NICE device protected human pluripotent stem cell-derived functional β cells (sc-β cells) from xeno-rejection and reversed diabetes up to 60 days in immunocompetent mice [34]. Compared to SC and epididymal fat pad (EFP) deployment, implantation in the IP space achieved a significantly thinner fibrotic layer and allowed laparoscopic device retrieval. However, IP implantation compromised vasculature development, which limited transplanted cells viability. To improve transplant viability, the same group developed an ‘inverse breathing’ encapsulation device, where CO₂ released from respiring cells is recycled into O₂ by lithium peroxide (Li₂O₂) particulates, in a self-regulated manner [35]. The SC implanted device uses silicon-based gas exchangeable materials to improve oxygen delivery to encapsulated islets, thus prolonging graft survival in multiple xenograft models [35]. However, these encapsulation devices require substantially more islets (>2-4 times) to achieve normal glycemia when compared to clinical intraportal pancreatic islet transplantation, which renders translation of these methods challenging.

The high metabolic and oxygen demands of cell transplants prompted a shift in the field, with most efforts focused on direct vascularization macroencapsulation technologies. As an example, devices such as Cell Pouch (Sernova) or Pec-Direct (Viacyte) were engineered to allow for direct vascularization into the device with the goal of improving engraftment and long-term function [29,36]. However, these approaches require systemic immunosuppression, which reverts to the problem of toxicity.

Evidently, achieving adequate graft oxygenation and metabolic sustenance for long-term survival with clinically feasible transplant volumes remains an unresolved challenge to clinical deployment of current encapsulation technologies.

3. Immunoengineering strategies for localized immunomodulation

The need for systemic immunosuppression in direct vascularization approaches motivated efforts to develop novel local immunomodulatory strategies. As key immune rejection events occur at the graft site, localized immunosuppression represents a rational, targeted approach of clinical relevance. The concept of local treatment of allografts to prevent immune rejection has been studied for a few decades [37–40]. Animal studies in the 1990s explored intra-arterial deployment of corticosteroids and CNI in allograft models to achieve localized delivery to cardiac and VCA grafts [37,38]. Two decades later, the feasibility of local immunosuppression via a biohybrid device for islet transplantation and central release of drug was studied for the first time [41]. Localized immunosuppression could attenuate inflammation within the graft environment to protect transplanted cells, sparing the patient from lifelong systemic immunosuppression and its associated complications. Specifically, engineering of biomaterials offers in situ immunomodulation opportunities through immobilization of surface ligands, local release of therapeutic agents through particles/scaffolds/hydrogels, and targeted delivery of nanocarriers [Table 1].

Table 1. Local delivery strategies used for therapeutic delivery of immunomodulation.

Therapeutic agents	Delivery strategy	Model	Ref(s)
Immune checkpoint molecules
FasL	Modified PEG microgels	Islet transplantation	[42]
	Modified PLG scaffold	Islet transplantation	[43]
PDL1	Modified PEG microgels	Islet transplantation	[45]
Anti-inflammatory cytokines
TGF-β1	Alginate scaffold	Allogeneic cell transplantation	[47]
	PLGA microparticles	Vascularized composite allotransplantation	[55]
	PEG-PLGA microparticles	Rheumatoid arthritis	[56]
	PLGA scaffolds/microparticles	Islet transplantation	[57,58]
IL-33	PLG scaffolds	Islet transplantation	[59]
IL-2 and TGF-β	Anti-CD2/CD4-coated PLGA nanoparticles	Systemic lupus erythematosus	[104]
T cell costimulation blockers
CTLA4Ig	Dual-reservoir encapsulation platform	Allogeneic cell transplantation	[68,159]
	ADM constructs	Islet transplantation	[69]
T cell depleting antibodies
αCD3	MECA79-coated nanoparticles	Heart allograft transplantation	[109]
Corticosteroids
Dex with E2	PDMS scaffolds	Host cell remodeling and vascularization	[89]
Dex	PLGA micelles	Islet transplantation	[90]
SIP receptor modulators
FTY720	PLGA films	Implant arteriogenesis	[96]
	PDMS scaffolds	Islet transplantation	[97]
	PHBV-PCL nanofiber constructs	Islet transplantation	[98]
mTOR inhibitors
Rapamycin	PS nanoparticles	Islet transplantation	[111]
	cRGD-coated PEG nanoparticles	Transplant vasculopathy	[112]
Anti-metabolites
Mycophenolate mofetil	PEG-PLGA nanoparticles	Transplant vasculopathy	[113]
Methotrexate	Composite hydrogel	Rheumatoid arthritis	[124]
Calcineurin inhibitors
Tacrolimus	TGMS hydrogel	Vascularized composite allotransplantation	[118]
	PLGA microparticles	Islet transplantation	[119]
	PLGA nanoparticles	Heart allograft transplantation	[107]
Cyclosporin A	PLA nanoparticles	Coupled delivery to lymph nodes	[108]
Other immunomodulators
Clodronate	Injectable matrigel	Islet transplantation	[121]
Clodronate with tacrolimus	Composite hydrogel	Islet transplantation	[122]

3.1. Surface functionalization of biomaterials

Surface functionalization entails the addition of a variety of factors to the surface of biomaterials, offering sustained presentation of immunomodulatory factors within the transplant microenvironment. As an example, engineered microgels and scaffolds with addition of streptavidin (SA) coupled with Fas ligand (FasL) allowed for controlled loading, presentation, and retention of SA-FasL protein within the graft microenvironment [42,43]. The Fas pathway is an immune checkpoint which inhibits uncontrolled inflammation [44]. In this context, microgels consisting of poly(ethylene glycol) (PEG) hydrogel beads modified with SA-FasL were co-transplanted with islets [42]. Alternatively, FasL-modified microporous scaffolds were loaded with islets [43]. In diabetic mice, both engineered biomaterials prolonged survival of allogeneic islets with median survival times of 31 or 46 days, compared to 14 days when transplanted with unmodified biomaterials [42,43]. Strikingly, the addition of a short course of rapamycin enhanced islet survival for over 200 days and restored euglycemia. Mice receiving the microgel engineered with SA-FasL had decreased numbers of CD4⁺ and CD8⁺ effector T cells (Teff) and increased numbers of Tregs at the graft site [42]. A similar approach using the PEG microgel platform leveraged programmed death ligand 1 (PDL1) [45]. The PDL1/PD1 costimulatory pathway inhibits alloimmune response and induces transplant tolerance. PEG microgels conjugated with SA-PDL1 for controlled delivery of PDL1 within the graft, in combination with a short course of systemic rapamycin, achieved survival of allogeneic islets up to 100 days. PDL1 delivery increased intragraft Tregs [45], consistent with the role of PDL1 as a Treg inductor [46].

An approach to promote in situ Treg differentiation was investigated via functionalization of TGF-β to alginate scaffolds [47]. When these scaffolds were co-transplanted with allogeneic fibroblasts, local generation of Foxp3⁺ Tregs was superior to constructs lacking TGF-β [47]. While functionalization approaches demonstrate potential efficacy to achieve local immunomodulation, a short course of systemic immunosuppression was still required to abrogate overall immune response and prevent allorejection [42,43,45]. These results indicate that a multi-targeted combinatorial approach affecting multiple pathways is necessary to achieve graft protection and prolonged function. Additional studies in large animal models are also necessary to demonstrate translatability.

3.2. Therapeutics with potential application for local immunomodulation

To date, there is no gold standard for immunosuppression after cell transplantation. Due to the complexity of the immune response toward allogeneic cells, clinical immunosuppression in a local setting is likely to require a multi-targeted, combinatorial drug approach. For pancreatic islet transplantation, current clinical protocols generally include systemic T cell depletion, a tumor necrosis factor α (TNF-α) blocker, tacrolimus, and sirolimus [48,49]. While local immunosuppression could subvert systemic toxicity, the effect of chronic local exposure of some agents on long-term cell viability and function remains detrimental. For example, the known toxicities of tacrolimus and sirolimus on pancreatic islet and renal cell viability, and impaired angiogenesis preclude their use in a localized setting [15,50–54]. Thus, elucidation of the safety profile of potential immunosuppressants is key for their future application in a localized setting. On that account, several other clinical immunosuppressants are appealing for use in a localized setting to prevent allograft rejection, improve graft survival through recruitment of anti-inflammatory cells, minimize toxicity associated adverse events, and improve overall patient quality of life [Figure 2]. We review a few of these promising agents as follows.

Figure 2. Therapeutics for local immunosuppression and sites of action. A) anti-CD3 binds to CD3 receptor on T cells. rATG binds to CD3. CD152, CD4, CD8, CD25 (IL-2 R) and CD28. CTLA4Ig binds to CD80 and CD86 on APCs, antagonizing CD28 costimulation. Anti-CD154(CD40L) disrupts CD40-CD40L costimulation. FTY720 engagement of S-1-P internalizes the receptors. B) rATG and anti-CD52 induce preferential inhibition of T cells. anti-CD52 and anti-CD3 trigger T cell anergy and apoptosis. APCs that engulf apoptotic vesicles from anergic T cells secrete TGF-β that promotes Treg and tolerogenic DC expansion. Anti-CD154 and CTL4Ig block T cell activation. Anti-CD154 and IL-33 promote a tolerogenic microenvironment. FTY720 induces vascularization and recruits anti-inflammatory myeloid cells.

3.2.1. Anti-inflammatory cytokines

Anti-inflammatory cytokines possess regenerative and immunoregulatory properties during inflammatory states. Namely, TGF-β has an essential role in inducing activated CD4⁺CD25⁻ cells to express Foxp3⁵⁴. In this context, encapsulation strategies for local TGF-β delivery were tested in models of allotransplantation and rheumatoid arthritis (RA) [55,56]. Intragraft TGF-β-loaded PLGA MPs failed to provide long-term allograft survival in a model of vascularized composite allotransplantation. However, graft survival was prolonged to >300 days when TGF-β was administered alongside IL-2 and rapamycin-loaded MPs [55]. Moreover, release of TGF-β1 from islet-containing PLGA scaffolds transplanted into the EFP, mitigated the immune response against allogeneic islets, decreasing production of inflammatory cytokines and leukocyte infiltration to the graft [57]. Similarly, co-delivery of TGF-β1 MPs and allogeneic islets within the same transplant site, showed minimal T cell infiltration in addition to increased Treg levels in the EFP proximal mesenteric lymph nodes when compared to controls [58]. However, local release of this monotherapy was insufficient to substantially delay graft rejection when compared to untreated controls [58]. Thus, a local multi-drug approach in combination with TGF-β1 is likely needed to induce long-term engraftment.

Localized delivery of IL-33, an M2-polarizing cytokine has been investigated for its immunomodulatory effects in adipose tissue [59]. IL-33 has both pro- and anti- inflammatory properties on alloimmune responses that drive graft-versus-host disease (GVHD). In addition, blocking the pro-inflammatory functions of IL-33 may enhance regulatory properties that contribute to improved transplant outcomes [60]. Loading IL-33 into islet containing PLG scaffolds implanted into the EFP, had a mixed inflammatory effect on graft acceptance in diabetic mice model [59]. Local IL-33 delivery resulted in local upregulation of Tregs, shifting their percentage of total CD4⁺ T cells from 40% to 75%. In addition, cytotoxic CD8⁺ T cells were downregulated, which prolonged graft survival from 14 to 33 days, compared to control scaffolds [59]. Even though transplanted islets showed extended survival, IL-33 delayed engraftment for 7 days. The delay in engraftment appears to be intrinsic to IL-33 given that a similar delay was also noted in syngeneic transplants used as controls [59]. IL-33 might induce innate cell populations that inhibit cell engraftment, although its mechanism remains unclear.

3.2.2. T cell costimulation blockers

Blockade of T-cell costimulation pathways allows modulation of adaptive immune responses that can suppress allograft rejection with less off-target effects than other immunosuppressive strategies [61]. CTLA4Ig is a clinically adopted fusion protein that binds to the B7 receptor on antigen presenting cells (APCs), effectively antagonizing CD28 costimulation and T cell activation [62], which can induce tolerance [63]. CTLA4Ig (belatacept) is clinically approved for kidney transplant rejection prophylaxis [64], and has shown success for islet transplantation in clinical trials [65]. CTLA4Ig in a local setting could circumvent toxicity due to systemic inhibition of normal immunoregulatory signaling of endogenous CTLA4 [66] and inhibit early events of alloimmune response [67]. In line with this, local delivery of CTLA4Ig demonstrated long-term allotransplanted islets survival and diabetes reversal in diabetic rodents [68,69]. Local delivery of CTL4Ig from human-derived acellular dermal matrix (ADM) constructs significantly prolonged islet allograft survival when implanted under the kidney capsule (KC). Co-transplantation of allogeneic islets in this matrix resulted in restored euglycemia for >150 days, in addition to Treg expansion and reduction in pro-inflammatory cytokines (IL-6 and IL-17) in serum [69].

Anti-CD154 (CD40L) disrupts the CD40-CD154 (CD40L) costimulatory pathway, halting pro-inflammatory cytokine production, Teff activation, and B-cell class switch, while promoting Treg expansion [70]. Unlike chemical immunosuppressants such as rapamycin and tacrolimus, anti-CD154 does not suppress graft function and prevents formation of alloantibodies and autoantibodies to the therapeutic monoclonal antibody [71]. Importantly, co-treatment with CTLA4Ig and anti-CD154 prolonged islet allograft survival due to the synergistic effect of blocking both costimulatory pathways [72–75]. The use of anti-CD154 is limited due to thromboembolic complications in clinical trials [76]; however, emerging second-generation agents that prevent thromboembolism while maintaining immunomodulatory action have been engineered [77]. We posit that these thromboembolic events and other complications could be avoided with local anti-CD154 delivery.

3.2.3. T cell depleting antibodies

rATG (or Thymoglobulin) is a lymphocyte-depleting, polyclonal antibody preparation, approved by the U.S. Food and Drug Administration (FDA) for induction immunosuppression in kidney transplantion [7,49]. rATG targets a myriad of T cell ligands including CD3, CD152, CD4, CD8, CD25, and CD28[78]. Clinically, rATG is administered transiently, as it induces profound immunosuppression with cytokine storm and increases infection risks [79]. Local dosing of rATG can obviate systemic toxicity associated with T cell depletion as well as prevent Teff cell activation or induce in situ T cell apoptosis [80].

Anti-CD3 is an FDA-approved immunosuppressant for cardiac and renal transplantation [81]. The main effect of anti-CD3 treatment is the induction of T cell anergy and apoptosis, with preferential depletion of conventional T cells while preserving Tregs [82]. Macrophages and APCs that internalize apoptotic vesicles from anergic T cells increase transforming growth factor (TGF-β) production which, in turn, promotes a tolerogenic microenvironment via Treg expansion and APC polarization into immunosuppressive phenotype [83]. The immunomodulatory effects of anti-CD3 on both T cells and APCs, support its use in a localized setting to promote immune tolerance at the graft microenvironment.

3.2.4. Corticosteroids

Anti-inflammatory glucocorticoids, such as dexamethasone (Dex), are often incorporated into biomaterial platforms due to their pleiotropic effects on the implant microenvironment [84–86]. Dex promotes anti-inflammatory macrophage phenotypes which in turn inhibit proinflammatory leukocytes [86,87]; however, it can also globally suppress cellular migration and delay vasculogenesis [88]. In an attempt to overcome the negative impacts of local delivery and assess effect on host cell remodeling, Dex was paired with proangiogenic 17β-estradiol (E2) in macroporous polydimethylsiloxane (PDMS) scaffolds [89]. Implantation of Dex-E2 scaffolds on the EFP of mice, resulted in decreased host cell infiltration and enhanced graft vascularization when compared to monotherapy Dex scaffolds after 14 days of implantation [89]. In another study, co-localization of Dex-loaded PLGA micelles and allogeneic islets under the KC, in addition to systemic CTLA4Ig therapy, led to long-term islet allograft survival and restoration of euglycemia in diabetic mice [90]. While the study is noteworthy, the KC is not a clinically relevant transplantation site. As such, further studies to validate the clinical translatability of results are warranted.

3.2.5. Sphingosine 1-phosphate receptor modulators

FTY720 is the first sphingosine 1-phosphate receptor-1 (S1P) agonist, a new class of FDA-approved immunomodulators for multiple sclerosis [91]. Fingolimod (FTY720) isolates lymphocytes in secondary lymphoid tissues, thus reducing circulation of T and B cells, effectively preventing alloimmune response [92]. Moreover, FTY720 tightens the endothelial barrier, preventing leukocyte transmigration to the graft [93]. Systemic toxicity of FTY720 is associated with the production of reactive oxygen species (ROS) [94], rendering local delivery an attractive approach [95]. Implantation of poly(lactic-co-glycolic acid) (PLGA) films loaded with FTYT20 in the site of injury, reduced proinflammatory cytokine secretion, and increased regenerative cytokines (IL-5, IL-10, and granulocyte colony-stimulating factor [GCSF]), leading to enhanced regeneration of ischemic tissues through recruitment of anti-inflammatory monocytes [96]. However, co-delivery of FTY720 with islets in macroporous PDMS scaffolds negatively impacted the cell engraftment in a dose-dependent associated cytotoxicity [97]. To address this, Bowers et al. [98] developed FTY720-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polycaprolactone (PCL) nanofiber constructs that surround a pocket formed by decellularized porcine dermis. Islets were loaded to the internal pocket of the construct via ultrasound-assisted imaging, 2 weeks after IP implantation. This preconditioning period established a regenerative microenvironment conducive to islet engraftment with clearance of FTY720 by the time of islet transplant, thus avoiding toxic effects.

Beyond inhibition of T cell activation, some drugs used for induction of anergy and management of early immune responses can promote Treg responses. The immunological impact of drugs like rATG and alemtuzumab (anti-CD52 mAb) is preferential inhibition of T and B cell activation over Treg expansion [99]. Specifically, rATG induction therapy reduces the absolute number of Tregs, favorably altering the Treg to T cell ratio [100]. Conversely, alemtuzumab can induce conversion of T cells to Tregs, increase anti-inflammatory cytokines IL-4, IL-10, and TGF-β, and suppress proinflammatory cytokines IFN-γ and IL-17 [99]. The need for preservation of Treg function concomitantly to the inhibition of T cell activation should therefore be considered of great relevance when selecting immunosuppressants in the context of transplantation. In addition, research is being conducted to induce immune tolerogenic microenvironments, as an alternative approach to immunosuppression. Wang et al. recently reviewed localized immunomodulation for transplantation of insulin-producing cells [101]. In summary, the opportunities deriving from the use of immunomodulatory agents in a local setting are largely unexplored, which provides a fertile field for scientific discoveries.

3.3. Targeted nanoparticles

Targeted delivery of drugs to lymph nodes has been explored for its potential to increase the efficacy of immune and cancer therapies while reducing associated toxicities [102,103]. Moreover, the ability of actively targeting encapsulated cargo to a subset of immune cells offers the opportunity to modulate the immune system without compromising systemic immunity [104]. Targeted therapeutic delivery through the use of nanoparticles (NPs) has recently been applied to provide tighter control over the delivery of immunomodulators in the context of allograft acceptance [105]. NPs can be engineered in size and properties that preferentially drain via the lymphatics to the lymph nodes, generating an efficient passive targeting strategy. Additionally, NPs may be actively targeted to immune-relevant sites via surface functionalization with selective ligands [Table 2].

Table 2. Targeted nanoparticle strategies for drug delivery and immunomodulation.

Therapeutic	Biomaterial	Modification (ligand, DC)	Target	Deployment	Model	Notable immunomodulatory effect	Ref
IL-2 and TGFβ	PLGA	Anti-CD2/CD4	T cells	IP	Sytemic lupus erythematosus	Increased circulating Tregs, suppressed anti-DNA antibody production	[104]
	PLGA	Anti-CD2	T cells	IP	Treg induction assessment	Increased frequency of Tregs on spleen and lymph nodes	[106]
Tacrolimus	PLGA	Passive targeting	Lymph nodes	SC	Heart allografttransplantation	Allograft survival for >28 days	[107]
Cyclosporin A	PLA	In vitrointernalization by DCs	Lymph nodes	SC	DCimmunotherapy	Efficient traficking of DCs to lymph nodes, suppression of CD8^+ T cells without systemic drug release	[108]
Anti-CD3	PEG-PLA	MECA79	PNAd molecules	IV	Heart allografttransplantation	Significantly prolonged allograft survival to >100 days	[109]
Rapamycin	PEG-b-PPS	In vivo internalization by DCs	Lymph nodes	SC	Islet transplantation	Significantly prolonged intraportal allograft survival to 100+ days, restored normoglycemia	[111]
	PEG	cRGD moieties	αVβ3integrins	Ex vivo	Allograft transplantation	Prevented transplant fibrosis and vasculopathy for >28 days	[112]
Mycophenolate mofetil	PEG-PLGA	Direct delivery	Allograft	Ex vivo	Transplant vasculopathy	Significant reduction in IL-6, TNF-α, IFN-γ and lymphocyte infiltration	[113]

Given the pathophysiology of transplant rejection, targeting NPs to lymph nodes and specific subsets of immune cells is an attractive immunomodulatory strategy. In a recent study, the surface of NPs was coated with anti-CD2/CD4 antibodies. These anti-CD2/CD4 PLGA NPs specifically target CD4⁺ T cells and deliver TGF-β and IL-2^104, [106]. After IP administration of CD4 targeted NPs, mice displayed increased Treg population frequencies in the mesenteric lymph nodes and spleen, whereas non-coated NPs at equivalent doses failed to achieve immune inhibition and tolerance [104,106]. In another approach, targeted release of TGF-β and IL-2 can increase local concentration of cytokines available to cells within nanoscale ligand-receptor distances, mitigating high dose-related toxicity while retaining high bioactivity [104]. Alternatively, tacrolimus was incorporated into PLGA NPs to prevent acute rejection after heart transplantation [107]. A single SC injection of tacrolimus-loaded NPs resulted in 50% of allograft survival over 28 days, achieving superior efficacy over intravenous (IV) and intragastric (IG) administration. This demonstrated that SC administration was the most efficient route to deliver PLGA NPs and enhance tacrolimus therapeutic effect. In another attempt to overcome the lack of selectivity and toxicity, targeted delivery of cyclosporin A (CsA) to lymph nodes was achieved by coupling CsA-loaded polylactic (PLA) NPs to allogeneic dendritic cells (DCs), which migrated from the SC injection site to draining lymph nodes [108]. This strategy aims to further improve the efficacy of DCs for immunotherapy by loading them with NPs that can carry different classes of drugs with no systemic release.

In efforts to target immune-cell-rich tissues, anti-CD3-loaded NPs were coated with MECA79 antibody which binds peripheral node addressin (PNAd) molecules, targeting them to peripheral lymph nodes [109]. The use of anti-CD3 in a transplantation setting is of interest, as it inhibits T cell expansion while inducing Tregs [110]. IV administration of MECA79-coated anti-CD3-loaded NPs significantly prolonged heart allograft survival to a median survival of >100 days, when compared to other forms of delivery [109]. This indicates efficient trafficking of MECA79-NPs to lymph nodes with subsequent release of anti-CD3. Furthermore, PEG-b-poly(propylene sulfide) (PEG-b-PPS) polymersome (PS) nanocarriers for SC delivery, significantly altered cellular biodistribution, redirecting rapamycins mechanism of action from immunosuppression to tolerance induction [111]. While standard oral rapamycin administration modulates T cells, rapamycin-loaded PS NPs delivered SC, drained into brachial lymph nodes where they were up-taken by APCs. As a result, APCs developed an anti-inflammatory phenotype with high MHCII for presentation to CD4⁺ T cell receptors, albeit without costimulatory molecules. Without costimulatory activation, acute rejection causes CD4⁺ T cells to become anergic or tolerogenic CD8⁺ Tregs [111]. SC delivery of rapamycin-loaded PS provided a tolerogenic state that allowed long-term survival of allogeneic islets transplanted intraportally in mice [111]. This study highlighted delivery of engineered NPs to repurpose drugs with known mechanisms of action and enhance therapeutic effect while mitigating adverse events.

Most of the studies investigating the use of NPs in transplantation have relied on systemic delivery with increased concentration in targeted sites. However, the treatment of donor organs prior to transplantation is a recently explored paradigm in transplant optimization [105,112]. Ex vivo incubation of mouse tracheal and aortic allografts with rapamycin-loaded PEG micellar NPs prevented transplant fibrosis and vasculopathy for 28 days post-transplantation [112]. Here, rapamycin NPs were coated with cyclic arginine-glycine-aspartate moieties (cRGD) specific for αVβ3 integrins located on the surface of endothelial and epithelial cells, thus, enhancing receptor-mediated endocytosis as compared to untargeted constructs [112]. Similarly, MMF was employed in a system of direct delivery and sustained release with NPs [113]. MMF is commonly used as a maintenance drug after renal transplantation as it inhibits inosine monophosphate dehydrogenase and blocks purine metabolism, preventing lymphocyte proliferation [114]. Perfusion of donor mouse hearts with MMF-loaded PEG-PLGA NPs prior to transplantation significantly reduced IL-6, TNF-α, and IFN-γ, as well as lymphocyte infiltration with intact vasculature in comparison to control and free MMF groups [113]. Altogether, these studies demonstrate that during standard procurement procedures, allografts can be treated with NPs prior to transplantation to reduce immune rejection and promote long-term survival.

Targeted drug delivery using NPs provides significant advantage over free-form administration by minimizing exposure of immunosuppressive agents to non-target tissues. However, despite increasing drug concentration at targeted sites, >95% of systemically administered NPs accumulate elsewhere in the body [115]. Moreover, nanoscale delivery systems are often susceptible to rapid clearance by the reticuloendothelial system (RES), further limiting their therapeutic effect [115]. Additionally, their use for chronic applications inevitably requires repeated administrations, which may pose concerns related to invasiveness of delivery, and poor adherence to treatment. These disadvantages have delayed translation of polymeric NPs into the clinic with only a small number being the FDA approved for cancer, iron-replacement therapies, delivery of imaging agents, fungal treatments, and macular degeneration. Nevertheless, numerous clinical trials are currently testing nanocarriers for cancer and gene therapy vaccines [116].

3.4. Hydrogel depot formulations

Another strategy for molecule-controlled release is using hydrogel systems as depot formulations to modulate the local microenvironment at the delivery site. Hydrogels possess excellent versatility in terms of providing multiple mechanisms for controlled drug release, as reviewed by Li and Mooney [117]. Only a limited number hydrogel drug delivery systems have reached clinical translation, with bone regeneration being the most impactful therapeutic application [117]. In these systems, controlled release of drugs is achieved through either network degradation, swelling, or mechanical deformation, which increase mesh size and promote outward drugs diffusion [117].

Localized immunosuppression with tacrolimus-loaded intragraft triglycerol monostearate (TGMS) hydrogels preserved function of allotransplanted hindlimb in rats for over 280 days without compromising kidney function, a hallmark side effect of tacrolimus therapy [118]. In a similar attempt, the SC co-delivery of tacrolimus-loaded PLGA MPs and pancreatic islets within Matrigel, effectively suppressed immune rejection by inhibiting T cell activation and macrophage proliferation in the local transplant microenvironment, restoring normoglycemia in diabetic mice [119]. In addition, the SC injected Matrigel may also provide an immune-protective barrier to transplanted islets and microspheres [119].

A recent study demonstrated effective IV delivery of immunosuppressive acid labile prodrugs targeted toward a SC injected azido-modified alginate gel depot [120]. In this study, prodrugs of tacrolimus, rapamycin, and MMF were designed to contain a clickable head, a water-soluble segment and a degradable linker responsive to lower pH indicative of inflammation. For the clickable head, dibenzocyclooctin (DBCO) was introduced as it can react with azido groups via Click chemistry. DBCO-modified prodrugs were successfully captured by azido-modified alginate hydrogels and cleared from the bloodstream after 12 h. This approach allowed IV deployment of fresh prodrugs into SC implanted hydrogels. Thus, refilling of hydrogel depots was achieved through noninvasive IV injection, which strategy could be used in other settings for drug supplementation once the payload is exhausted. However, efficacy demonstration of this approach for allotransplantation remains to be elucidated.

Induction of tolerance to islet allografts through local delivery of immunomodulators with hydrogels has been explored [121,122]. Injectable hydrogel Matrigel was used for local delivery of immunomodulatory liposomal clodronate in conjunction with pancreatic islets [121]. Clodronate causes macrophage depletion and decreases expression of adhesion molecules in the synovial lining of patients with longstanding RA [123]. In particular, the protection imparted by clodronate after islet transplantation was key for long-term survival of islets (>60 days) [121]. Local release of clodronate lowered concentration of proinflammatory cytokines in the islet-containing Matrigel [121]. In a similar attempt, Pathak et al. explored local co-delivery of clodronate and tacrolimus with a composite hydrogel using MPs embedded in Matrigel to induce tolerogenic DCs and protect xenogeneic islets from rejection [122]. Local SC co-delivery of islets and immunosuppressants embedded in the Matrigel resulted in decreased expression of CD40, CD80, CD86, and MHCII, characteristic of a tolerogenic DC phenotype, in both the graft and draining lymph nodes. Consequently, after tolerogenic induction and subsequent secretion of TGF-β and IL-10 by tolerogenic DCs, Tregs were increased in the spleen, draining lymph nodes, and graft [122]. Similarly, local delivery of indomethacin and methotrexate was tested using a composite system of polyethyleneimine NPs within a Pluronic hydrogel in a rodent model of RA [124]. After intra-articular injection, controlled release of drugs was prolonged with slower release rates in the composite hydrogel system as compared to NPs alone [124]. These results highlight composite hydrogels as a strategy to develop local and controlled-release systems through addition of secondary barriers to regulate drug diffusion.

4. Co-transplantation with immunomodulatory cells and extracellular vesicles

Tolerance induction protocols employing co-transplantation of allogeneic cells with immunomodulatory cells have the potential to reduce maintenance immunosuppression regimens. We review a few of these graft supportive cells as follows.

4.1. Co-delivery with mesenchymal stem cells (MSCs)

MSCs are multipotent stem cells widely investigated for their pivotal role in vascular regeneration and immunomodulation [125]. The co-delivery of MSCs promotes graft survival and tolerance induction to third-party cardiac [126], kidney [127], endothelial cell induced vascular structure [128], corneal [129], and islet [130] allografts. These graft-beneficial effects can be attributed to the ability of MSCs to induce a pro-tolerogenic Treg phenotype [126,127,129] and reduce the inflammatory alloimmune responses [128]. Although MSCs co-transplantation delays allograft rejection, survival beyond 100 days has yet to be achieved.

To achieve tolerance induction with cell co-delivery strategies, bone marrow-derived MSCs (BM-MSCs) are preclinically and clinically studied for co-transplantation with islets [131,132]. The source of MSC plays a key role in the outcome of graft survival. In fact, co-transplantation of autologous BM-MSCs protected against alloimmune response and prolonged intrahepatic islet engraftment [131]. A randomized controlled trial by Tan et al. [133] demonstrated that treatment with autologous BM-MSCs as induction therapy, IV infused at day 0 and 14 after kidney transplantation, reduced CNI immunosuppression requirement. Additionally, when compared with anti-IL-2 receptor antibody induction therapy, autologous BM-MSCs decreased incidence of acute graft rejection and risk for opportunistic infection, improving estimated renal function at 1-year follow-up[133]. Further, a recent case provided evidence that infusion of autologous BM-MSCs was associated with safe complete discontinuation of maintenance immunosuppression after renal transplantation, allowing a state of immune tolerance [134]. Overall, preclinical models have shown the feasibility of effective MSC therapy in transplantation with early clinical trials supportive of their safety.

4.2. Co-delivery with myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immunosuppressive cells from the myeloid lineage, characterized by expression of CD11b, CD33, CD34, and vascular endothelial factor (VEGF) receptor 1[135]. MDSCs are upregulated by mediators of inflammation such as IL-2, GCSF, TGF-β, and PGE2[136]. Upon exposure to stimulatory molecules, MDSCs can suppress T cells and promote transplant tolerance via intragraft Treg expansion [137]. Studies have demonstrated that MDSCs secrete IL-10, CTLA4 and IFN-γ to increase the number of Foxp3⁺ Tregs. Treg functions are upregulated by the interaction of PDL1 on MDSCs with PD1 on Tregs [138]. MDSCs are shown to prolong graft survival in corneal [139], islet [140], cardiac [141], kidney [142], and skin [143] transplantation models and induce transplant tolerance. However, the effector function of MDSCs after transplantation remains poorly understood. Transwell assays showed that MDSCs produce soluble TGF-β to modulate T cells, as well as ROS to regulate Th2 immune responses [138]. Induction of transplant tolerance using clinical immunosuppressants combined with the immunomodulatory effects of MDSCs in a local setting warrants further investigation.

4.3. Co-delivery with regulatory T cells

Tregs are a unique subset of immune cells (CD4⁺CD25⁺Foxp3⁺) that play an important role in maintaining homeostasis and tolerance. Tregs suppress immune responses through DC modulation, inhibitory cytokine secretion, and cytolysis and metabolic disruption of Teffs [19]. Over the last years, some Treg-based cell therapies have reached clinical trials [19]. Treg cell therapies reduced requirement of induction immunosuppression for renal transplantation and maintenance immunosuppression for liver transplantation in phase I/II clinical trials [144,145]. Although still in the early stages, preliminary results from these trials showed significant reduction of infection rates in patients [144]. These results underscore Treg therapy potential to minimize the burden of systemic immunosuppression in transplant patients.

4.4. Cell-free immunomodulation with extracellular vesicles

Extracellular vesicles (EVs) produced by both immune and non-immune cells have an important role in the regulation of immunity. EVs possess both immunostimulatory and immunosuppressive properties, and therefore can be employed as immunomodulatory agents for transplantation [146]. Administration of DC-derived EVs before and after transplantation promoted overall immune tolerance to kidney allografts [147]. Use of immature DC-derived exosomes significantly improved graft survival and reduced inflammatory response by promoting Tregs and limiting CD4⁺ T cell infiltration [147].

Similar to MSCs, MSC-derived EVs are capable of modulating T cell functions and immune responses. Recent efforts have focused on the therapeutic effects of MSCs through the secretion of paracrine factors, instead of engraftment and differentiation into functional cells [148]. In vivo studies with MSC-derived EVs showcase their potential to be used as alternative to MSC-based therapies [148]. MSC-derived EVs possess immunoregulatory properties that prevent acute graft-versus-host-disease (aGVHD) [149–151]. IV administration of BM-MSC-EVs in previously induced aGVHD mice resulted in a temporary increase of circulating Tregs as well as levels of TNF-α and IFN-γ in plasma, peaking at day 9 after administration and prolonging mice survival until experimental endpoint of 30 days, whereas untreated mice did not survive beyond day 15¹⁴⁹. In another study, aGVHD mice that received systemic infusion of EVs showed improvement with suppression of GVHD-associated pathology in the colon, particularly of inflammatory cell infiltration to the lamina propia [150]. Moreover, EVs derived from umbilical cord-derived MSCs (UC-MSCs) reduced circulating CD8⁺ T cells as well as serum levels of IL-2, TNF-α, and IFN-γ after IV administration to recipient mice receiving allogeneic HSCs for aGVHD induction [151].

In comparison to whole cell-based therapies, MSC-EVs employed as cell-free therapies may offer specific advantages for patient safety such as lower propensity to trigger immune responses and inability to form tumors [152]. Moreover, different methods for preservation of EVs are under investigation to extend their shelf life and reduce storage-related costs compared to cell-based therapies [153]. However, the characterization and application of MSC-derived EVs is limited by low-yield and labor-intensive isolation techniques, as well as lack of standardized isolation protocols [149]. In fact, MSC-derived EVs paracrine factor content greatly varies depending on the techniques used for isolation and the activation status of MSCs [149]. Currently, EVs have yet to be assessed in clinical trials, although a number of studies have examined the effect of vesicles on isolated human organs, demonstrating a potential reparative effect [154].

5. Other emerging strategies for local immunomodulation

Although the transplantation of immunoprotected therapeutic cells may provide a translatable solution, the lack of blood supply to the microenvironment hinders adequate graft oxygenation. In an effort to achieve adequate graft oxygenation, prevascularization was induced using MSCs prior to allogeneic transplantation in cell encapsulation macrodevices [155–157]. In this context, to eliminate the adverse effects of systemic immunosuppression while preserving the advantages of direct graft vascularization, Paez et al. developed a neovascularized implantable cell homing and encapsulation (NICHE) platform [68,158]. The NICHE is a transcutaneously replenishable, dual-reservoir implantable device that integrates direct vascularization and localized delivery of soluble immunosuppressants [Figure 3A] [68,155]. Two independent drug and cell reservoirs are interconnected by a nanoporous membrane that enables uninterrupted and controlled immunosuppressant elution for months. The NICHE deployment strategy consists of a prevascularization phase after SC implantation with MSCs, followed by transcutaneous loading of immunosuppressants and therapeutic cells into the drug and cell reservoirs, respectively [68]. Local release of CTLA4Ig immunosuppressant maintained allogeneic Leydig cell viability in immunocompetent rats, with low lymphocyte infiltration within the NICHE. Importantly, confinement of immunosuppressant to the transplant microenvironment limited systemic exposure while maintaining allogeneic cell survival up to 31 days [68]. Furthermore, NICHE efficacy for allotransplantation of islets and long-term diabetes reversal was demonstrated in an immunocompetent rat model [159]. Transplantation of allogeneic islets within prevascularized NICHE restored normoglycemia in rats for >150 days and local immunosuppressant delivery prevented immune rejection without inducing systemic immunosuppression. Specifically, local anti-lymphocyte serum (ALS) and CTLA4Ig delivery reduced immune cell infiltration and prolonged graft survival similarly to standard systemic delivery, albeit with unchanged circulating T cell and Treg levels. Moreover, for translatability efforts, ease for deployment and short-term allogeneic islet engraftment were demonstrated in a fully MHC-mismatched primate model. Partial immunosuppression was achieved with addition of anti-CD154 to the immunosuppressive cocktail, effectively reducing leukocyte infiltration in fully vascularized devices and localized drug delivery abrogating systemic drug exposure.

Figure 3. Other emerging strategies to overcome systemic immunosuppression in cell transplantation. A) The neovascularized implantable cell homing and encapsulation (NICHE) platform integrates localized delivery of immunosuppressants and direct vascularization. The drug and cell reservoirs are interconnected via nanoporous membranes for controlled release of immunosuppressants into the cell reservoir providing local protection of allogeneic cells against immune cells. Self-sealing silicone ports allow minimally invasive transcutaneous refilling of drug and cell reservoirs. B) Upregulation of CD47 via lentiviral transduction and knock-out (KO) of MHCI and MHCII via CRISPR-Cas9 technology, creates hypoimmunogenic induced pluripotent stem cells (iPSCs) that do not elicit immune responses in allogeneic recipients.

An alternative approach entails engineering hypoimmunogenic pluripotent stem cells as a source of universally compatible cell or tissue grafts. This concept stemmed from drawbacks in pluripotent stem cell reprogramming [160]. Reprogramming technologies allowed the generation of autologous human induced pluripotent stem cells (hiPSCs) for patient-specific treatments. However, hiPSCs production is laborious, costly and is only practical for certain conditions [160]. Instead, the use of allogeneic stem cell therapies can target large patient populations but would require strong immunosuppression. For these reasons, research has focused on generating hypoimmunogenic cell lines that allow transplantation to multiple recipients, while attenuating immune rejection response. Deuse et al. [160] found that syncytiotrophoblast cells, which are intrinsically hypoimmunogenic due to contact between maternal blood and fetal tissue, have downregulation of MHCI and MHCII and upregulation of CD47 molecules. This led to the design of hiPSCs with MHCI and MHCII knockout (KO) using CRISPR-Cas9 technology via the B2M and CIITA gene, and lentiviral transduction for upregulation of CD47 [Figure 3B]. These gene-edited hiPSCs did not induce an immune response in HLA mismatched allogeneic humanized mice recipients. Moreover, differentiation of hiPSCs into cardiomyocytes and epithelial cells did not cause immune rejection throughout the 50 days study duration [160].

As an innovative solution to donor organ shortage, gene editing via CRISPR-Cas9 technology has been leveraged to reduce immunogenicity of porcine tissues, making xenotransplantation a potentially feasible solution [161,162]. Yang et al. reported up to 65 simultaneous edits allowing the removal of all copies of the porcine endogenous retrovirus (PERV) pol gene from the pig genome, rendering them more compatible with humans [161,162]. The long-term goal is to produce universal donor cell lines for replacement therapies as a solution to organ shortages, with efforts to advance xenotransplantation into clinical trials [163].

6. Conclusion

Emerging local immunoengineering strategies have the potential to significantly enhance cell transplantation outcomes. However, a great overarching challenge in cell allotransplantation remains overcoming the complexity of the immune response. Addressing knowledge gaps in the crosstalk mechanisms that occur during graft rejection as well as developing complementary and synergistic approaches that target multiple components of the alloimmune response is crucial to achieve long-term protection of transplanted cells without the risk of systemic adverse events.

7. Expert opinion

Cell transplantation is undergoing an important transition from the traditional approach that targets systemic immune responses to more effective and less toxic methods of immune modulation at the local graft microenvironment. While cell encapsulations that provide a physical barrier against immune rejection have been extensively explored to avoid systemic immunosuppression, lack of direct graft vascularization precluded their successful development. Subsequent efforts were made to achieve graft oxygenation reviving the idea of using cell replacement therapy in conjunction with a method aimed to deter immune cells from attacking the grafted area.

Advances in bio- and immunoengineering have spurred tailored approaches to address these challenges. Specifically, biomaterials and surface functionalization provide versatile tools to locally modulate the immune response, while stimulating the growth of a functional vasculature network. It is at the graft microenvironment that nanocarriers, scaffolds, and hydrogels can be adapted for local and personalized release of immunomodulatory agents directed against acute and chronic transplant rejection.

In this context, local delivery of immunomodulatory agents presents a unique opportunity to address the limitations of systemic delivery avoiding toxicity, rapid clearance, and off-target effects. For this, while biomaterials functionalization can provide transient therapeutic efficacy that is relevant in the short term against chronic rejection, platforms that allow for sustained, and potentially life-long immunomodulation are needed for extending life of the transplant. Here, simple mechanisms for drug refilling or supplementation may be fundamental for long-term clinical deployment. Nonetheless, it is still unclear if local therapeutic release is sufficient to regulate the alloimmune response. In addition, local confinement of immunosuppressants impose potential toxicity to the graft. Thus, safety profiles for local drug release are required as with systemically deployed agents.

Additionally, local immunomodulation opens the door to new developments in immunotherapeutic agents. Designed to achieve antigen-specific tolerance and maintenance of Tregs as opposed to immunosuppression, these may shorten the course of anti-rejection therapy while achieving long-term graft viability. This could be achieved in concert with providing secondary immune modulation through cells such as MSCs, which provide the distinct advantage of physiological and continuous production of cytokines to modulate the immune microenvironment. Alternatively, sub-cellular components such as extracellular vesicles could be used aiming at reducing activation of T and B cells. The benefits could be multiple, including improving adherence to treatment, limiting treatment fatigue, and visits to the healthcare centers, and improvement of patient quality of life.

It must be noted, however, that the solution to long-term successful cell transplantation may require a multipronged approach including biomaterials, cells, and therapeutics, which is likely to render the clinical translation complex. As an example, the process of producing therapeutic cells to meet clinical standards is cumbersome, expensive, and complex in itself. To this end, the prospected use of engineered hypoimmunogenic cells is attractive as it does not require third-party biomaterials or supporting cells; however, the genetic modifications to achieve cell immortalization could compromise the long-term cell physiology. Engineering universal cells that can evade immune rejection hold great potential in overcoming donor shortage; however, despite the advances in genomic editing technologies, a risk of developing genomic instability over time still exists. Moreover, the idea of having cells highly capable of evading the immune system impose safety concerns for clinical applications.

Novel immunoengineering strategies have set a new standard in the field of transplantation, and we believe that future immunosuppression protocols should shift from systemic to local immune modulating strategies. Shifting to local delivery systems that allow multi-targeted strategies to modulate the immune response in the transplant microenvironment will address the limitations of current encapsulation technologies for clinical cell transplantation. Promoting these new methodologies will ultimately lead to clinically significant increases in treatment quality resulting in overall improved patient care.

Article highlights

• The requirement of systemic immunosuppression to prevent transplant rejection is a major hurdle in allotransplantation.

• Novel immunoengineering strategies offer a unique opportunity to prevent transplant rejection without the adverse events of systemic immunosuppression regimens.

• Localized immunosuppression represents a targeted approach to tackle immune rejection specifically at the graft site.

• Local immunomodulation is designed to achieve antigen-specific tolerance and maintenance of Tregs as opposed to immunosuppression.

• The future of transplant rejection lies in the shift from systemic to local immune modulating strategies to promote transplant tolerance in a safe an effective manner.

Declaration of interest

J Paez-Mayorga, C Ying Xuan Chua, and A Grattoni are inventors of intellectual properties licensed by NanoGland, LLC. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was funded by the Juvenile Diabetes Research Foundation (JDRF 2-SRA-2021-1078-S-B) and Vivian L. Smith Foundation.