Harnessing the potential of red blood cells in immunotherapy

Abstract

Red blood cell (RBC) transfusion represents one of the earliest and most widespread forms of cellular therapy. While the primary purpose of RBC transfusions is to enhance the oxygen-carrying capacity of the recipient, RBCs also possess unique properties that make them attractive vehicles for inducing antigen-specific immune tolerance. Preclinical studies have demonstrated that RBC transfusion alone, in the absence of inflammatory stimuli, often fails to elicit detectable alloantibody formation against model RBC antigens. Several studies also suggest that RBC transfusion without inflammation may not only fail to generate a detectable alloantibody response but can also induce a state of antigen-specific non-responsiveness, a phenomenon potentially influenced by the density of the corresponding RBC alloantigen. The unique properties of RBCs, including their inability to divide and their stable surface antigen expression, make them attractive platforms for displaying exogenous antigens with the goal of leveraging their ability to induce antigen-specific non-responsiveness. This could facilitate antigen presentation to the host’s immune system without triggering innate immune activation, potentially enabling the induction of antigen-specific tolerance for therapeutic applications in autoimmune disorders, preventing immune responses against protein therapeutics, or reducing alloreactivity in the setting of transfusion and transplantation.

1. Introduction

Cellular therapies represent a rapidly evolving field in medicine, offering promising new approaches for treating various diseases, including cancer, autoimmunity, and degenerative conditions [1]. These therapies are characterized by the use of cells as therapeutic agents, wherein the intrinsic and dynamic capacity of a cell to regulate an entire range of responses can be harnessed to facilitate desired clinical outcomes.

A variety of cellular therapies have emerged in the last few decades. Among these, the ability to reprogram T cells, often in the form of chimeric antigen receptor (CAR) T-cell therapy, reflects an increasingly common strategy that continues to completely change cancer treatment [2]. Similar approaches are being used to more effectively eliminate immune populations that may contribute to autoimmunity [3]. Stem cell therapies, which often seek to harness the ability of stem cells to differentiate into distinct cell states, may also hold promise in the treatment of autoimmunity, transplant rejection and regenerative medicine. Mesenchymal stem cells in particular have been shown to promote tissue repair and favorably modulate patient immune function [4]. Additional studies are being leveraged to enhance the delivery of these therapies, including novel strategies aimed at endowing various forms of cellular therapies with distinct molecular signatures that enhance homing to desired tissue [5].

While these new therapies hold promise in transforming modern medicine, the earliest and most common form of cellular therapy continues to be simple blood transfusion. The transfusion of blood products, which represents the most common intervention in hospitalized patients, is a mainstay of clinical care for a wide variety of patients, including patients with neoplastic disease, those that require surgery and patients with underlying transfusion-dependent conditions [6]. Transfusion therapy can reflect the delivery of whole blood or individual components in the form of red blood cells (RBCs), platelets, and plasma. Transfusion of blood products can represent a life-saving intervention in hemorrhaging patients, critical support following introduction of chemotherapy or bone marrow transplantation and a necessary long-term intervention in patients with underlying conditions that impact RBC efficacy. Plasma derivatives, such as albumin and clotting factor concentrates, are also used for a variety of patient populations, although the collection of plasma and the manufacturing process required to generate these products typically occurs through processes distinct from routine blood donation that can make these products uniquely vulnerable to changes in blood donation trends [7].

1.1. Blood transfusion as a personalized therapy

In contrast to the personalized approaches to many forms of next generation cellular therapies, wherein products are often specifically modified to enhance their ability to target unique features of a given disease, blood transfusion typically employs blood cells in their existing state, yet requires a personalized approach to prevent alloantibody reactivity that can lead to hemolytic transfusion reactions [8 9 10]. Early attempts at blood transfusions often led to variable and sometimes fatal outcomes due to lack of understanding about blood group incompatibility. It wasn’t until Landsteiner’s discovery of ABO blood group antigens that the underlying etiology of differences in transfusion outcomes became apparent and a possible way to predict transfusion outcomes was realized. The discovery of the ABO blood group systems resulted in the first description of a polymorphism in the human population, which would subsequently be recognized to have significant implications in human evolution, risk for infectious disease transmission and overall cardiovascular health [9 11 12 13 14 15 16]. With respect to transfusion, however, this discovery has allowed pre-transfusion testing to identify compatible blood types prior to transfusion, dramatically reducing the risk of life-threatening hemolytic reactions [17]. In doing so, the ability to match donor and recipient blood types made transfusion medicine possible and ultimately paved the way for organ transplantation.

Following the implementation of blood transfusion, alloimmune incompatibilities beyond ABO antigens that can likewise cause incompatibility and induce fatal hemolytic transfusion reactions were discovered. However, unlike anti-ABO(H) antibodies, which form within the first few months of life without known exposure to allogeneic RBCs, most non-ABO alloantibodies form in response to allogeneic RBC exposure as a result of pregnancy or transfusion [8]. Alloantibodies that form in this setting can not only become a barrier to safe transfusion but can also cross the placenta and attack fetal red blood cells during pregnancy, resulting in hemolytic disease of the fetus and newborn. There have been over 300 blood group antigens described against which individuals can develop alloantibodies [10]. Prior to blood transfusion, in addition to testing for ABO blood groups and corresponding anti-ABO(H) blood group antibodies, an alloantibody screen is performed to determine whether a given patient has developed alloantibodies against any of these other alloantigens [8].

For most patients requiring blood transfusion, identifying compatible RBCs is not challenging. Adequate testing of ABO(H) blood group antigens allows compatible RBCs to be obtained for transfusion. However, for patients who have generated alloantibodies against a variety of blood group antigens or against an alloantigen that has high prevalence within the blood donor population, the ability to find compatible blood, especially in urgent situations, can be difficult [18 19]. In these patients, a combination of anti-ABO(H) and non-ABO(H) alloantibodies can make it difficult to find RBC units that are safe for transfusion [20]. This can be especially problematic in transfusion-dependent patient populations, such as patients with sickle cell disease (SCD) [21 22 23 24]. Owing in part to challenges associated with timely procurement of blood and complications that can result from the development of alloantibodies, recent studies have demonstrated that alloimmunized patients can experience higher mortality and increased morbidity [25 26].

Even when alloantibodies are not detected at the time of transfusion, previous alloimmunization can place patients at risk for developing delayed hemolytic transfusion reactions (DHTRs). Variability in alloantibody persistence makes it difficult to consistently detect alloantibodies. This places patients at risk for accelerated RBC removal following subsequent transfusion and the development of life-threatening DHTRs [27 28 29 30 31 32]. Once thought to be rare [33], these reactions can occur in as frequently as 1/25 episodic transfusions in patients with SCD with a case fatality rate of 11 %, making these reactions the most common and severe transfusion complications in this patient population [21 27 34]. To actively prevent all forms of RBC alloimmunization, the unique immune pathways that drive distinct antibody outcomes must be defined. As transfusion-dependent patients live longer, the challenges surrounding the development and consequences of RBC alloimmunization have become more pronounced [20].

1.2. Addressing the challenges of RBC alloimmunization

In an effort to address the challenges associated with RBC alloimmunization, especially in transfusion-dependent patients, a variety of epidemiological studies have been conducted to understand risk factors that may influence the likelihood of alloantibody development following RBC transfusion [35 36 37 38 39 40 41 42 43 44 45 46 47 48]. These studies suggest that viral infection at the time of transfusion, chronic disease states (such as sickle cell disease and myelodysplastic syndrome), prior alloimmune responses, and certain genetic polymorphisms can increase the likelihood of RBC alloimmunization, while prior splenectomy, gram negative infection at the time of transfusion and alternative genetic polymorphisms may reduce RBC alloimmunization [35 36 37 38 39 40 41 42 43 44 45 46 47 48].

While patients with SCD have been shown to have higher alloimmunization rates, the indication for transfusion can have a significant impact on whether RBC alloimmunization occurs. Transfusion of patients with SCD for stroke results in an alloimmunization rate that is comparable to the general population. Consistent with this, no difference in the underlying response to vaccination can be observed when patients are stratified based on RBC alloantibody responder status [49]. In contrast, transfusion during acute chest syndrome (ACS) significantly increases the risks of alloimmunization (odds ratio 16.7; 95 % CI 10.7–26.4) [50 51 52 53 54]. While further research is needed to elucidate the impact of ACS on RBC alloantibody formation, elevated levels of heme, often observed during complications necessitating transfusion [55 56 57 58 59 60 61 62 63], can modulate the activity of various immune cells implicated in the alloimmune response to transfusion, including monocytes, dendritic cells, B cells, and T cells. These findings suggest that increased heme concentrations may influence an individual’s propensity to mount an immune response against transfused RBCs. Consistent with this, studies have shown that heme appears to suppress the function of these immune populations in transfused patients who failed to generate alloantibodies, while heme failed to similarly suppress immune activity among alloimmunized patients [64 65 66]. Consequently, heme responsiveness may shape the predisposition of SCD patients to develop alloantibodies after transfusion. Distinct baseline characteristics of immune cell subsets, such as monocytes and T follicular helper cells, have also been observed in individuals more prone to alloimmunization compared to those who remain non-alloimmunized despite exposure to RBC alloantigens [44 67]. Differences in interferon signatures may also predict alloimmune responses [68]. These intrinsic differences, coupled with the immunomodulatory effects of heme, may contribute to the variable alloimmunization rates observed among transfused SCD patients [69 64 65 66].

2. Studying alloimmunity uncovered opportunities to use RBCs as vehicles of tolerance

While epidemiology studies have provided important insight into factors that can influence RBC alloimmunization, it has historically been difficult to pinpoint how the immune response to RBC transfusion initially develops following RBC alloantigen exposure at a mechanistic level. As only ABO(H) and RhD are matched for clinically in the emergent setting, it is not ethical to deliberately induce RBC alloantibodies in patients against other alloantigens experimentally as this would put patients at risk for an acute hemolytic transfusion reaction should emergent transfusion be required. Even if intentional alloantigen exposure was ethically permissible, the low per-unit alloimmunization rates for most RBC antigens make studying this process extremely challenging in patient populations. Furthermore, key features of immunity to alloantigens require trafficking of immune cells outside of blood into tissues [70 71 72]. As a result, the immune cells and the events they govern are difficult to study in peripheral blood alone. This is especially important when considering that host immune tissue architecture is critical in governing immune function [70 71 72].

Given constraints in conducting alloimmunization studies in humans, preclinical animal models have become complementary tools in efforts to delineate the key immune mechanisms governing RBC alloantibody responses [50 73 74 75 76 77 78 79 80] ( Table 1 ). Early investigations in murine models revealed that RBC transfusion alone, in the absence of inflammatory stimuli, often fails to elicit detectable alloantibody formation against model RBC antigens. This mirrors the clinical observation where most transfusion recipients remain “non-responders” and do not generate detectable alloantibodies despite repeated exposures to allogeneic RBCs. Indeed, while chronically transfused patients are often categorized as responders or non-responders based on the presence or absence of detectable alloantibodies, the majority of patients fall into the latter category on a per transfusion basis and do not mount a measurable alloimmune response following a given RBC transfusion [81].

Table 1.

Preclinical models for RBC alloimmunization.

RBC model	Requires MZ B cells	Requires CD4 T cells (IgG)	Regulated by complement	Enhanced by storage	Regulated by TLRs	Antigen specific non-responsiveness
mHEL *	NT **	NA ***	NT	NT	NT	Y
GPA	NT	NA	NT	NT	NT	Y
HOD	Y	Y	N	Y	Y	N
KEL hi	NT	NA	NT	NT	NT	Y
KEL	Y	N	Y	N	NT	N
KEL lo	NT	NA	NT	NT	NT	Y

^*mHEL=membrane bound HEL, GPA=glycophorin A, HOD=HEL, OVA and Duffy, TLR=toll-like receptors.

^**Y=Yes, N=no, NT=not tested, NA=not applicable, V=variable.

^***IgG response in the presence of viral-like inflammation can involve CD4 T cells.

While transfusion often fails to induce alloimmunization, numerous studies across multiple preclinical models have highlighted the critical role of recipient inflammation in dictating the immune response to transfused RBC alloantigens. In the absence of an inflammatory stimulus, RBC transfusion alone often fails to elicit detectable alloantibody formation, mirroring the clinical observation where most patients remain non-responders despite repeated alloantigen exposures. However, recipients can become responsive if allogeneic RBCs are transfused in the presence of viral-like substances capable of triggering innate immune pathways. Viral mimics like polyinosinic:polycytidylic acid (poly I:C), intact viruses or disease conditions associated with elevated type I interferons have been shown to significantly enhance RBC alloimmunization in murine models by promoting antigen consumption by dendritic cells (DCs) and increasing co-stimulatory molecule expression [51 82 83 84 85]. Conversely, gram-negative bacterial products may impair alloantibody responses, suggesting distinct effects of different inflammatory stimuli through the differential alteration of sentinel immune cells that govern alloimmune responses [83]. In contrast, if a recipient is exposed to some RBC alloantigens in the absence of inflammation, these transfused recipients can become unresponsive to the same antigen even upon subsequent transfusion in the presence of inflammation [74 76 86]. This phenomenon implies that the initial context of antigen encounter is not without consequence and instead possesses the ability to influence host immunity in an antigen specific manner.

The mechanisms underlying the impact on inflammation on recipient alloimmunization following transfusion likely involves a variety of immune cells, including modulation of antigen-presenting cell (APC) function and the inflammatory milieu. Inflammation can shift RBC consumption towards more immunogenic DC subsets, while also providing requisite danger signals for DC maturation and effective antigen presentation to T cells [87 88]. Additionally, studies suggest that distinct innate immune sensors may be differentially engaged by various inflammatory stimuli, impacting downstream adaptive immunity. For instance, type I interferon signaling has been implicated as a key mediator of viral-enhanced RBC alloimmunization [89 90]. Recent studies elegantly demonstrate that increased numbers of reticulocytes may likewise trigger innate immune pathways possibly through enhancing exposure to retained mitochondria [91]. Overall, these findings underscore the intricate interplay between inflammatory cues, innate immune sensing, and the delicate balance between tolerance and immunity in the context of RBC alloantigen exposure. Elucidating these pathways holds promise for developing strategies to modulate alloimmunization risks in transfusion recipients.

While inflammatory stimuli triggered by viral mimics, increased numbers of reticulocytes or even factors released during blood storage can potently augment RBC alloimmunization [91 92 93 94], alloantibody development can also occur in the absence of exogenous adjuvants, unlike common pathways thought to be required for immune responses to effectively occur during infection. This raises intriguing questions regarding the specific immune cell populations capable of sensing and responding to transfused RBC alloantigens in the absence of a known adjuvant, and how these cells may be uniquely poised to receive tolerogenic or immunogenic signals that ultimately dictate the outcome of RBC antigen exposure. Recent evidence suggests that transfused RBCs localize to the marginal zone (MZ) in the spleen, where they can be found to co-localized with MZ B cells and marginal zone macrophages (MZMs), two specialized immune sentinels residing in the marginal sinus of the spleen [72 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109]. MZ B cells are an innate-like B cell population that is uniquely interfaced at the blood-lymphoid compartment along the marginal sinus and are therefore ideally suited to rapidly capture and respond to blood-borne RBC alloantigens. Several studies have demonstrated that MZ B cells can initiate alloantibody formation against several model RBC alloantigens [95 97] ( Fig. 1 ). Intriguingly, MZ B cells have also been implicated in initiating alloimmune responses against other blood-borne antigens, including platelets and factor VIII [110 111 112], suggesting a broader role in surveying the blood compartment. Subsequent studies suggest that macrophages in the marginal zone may likewise be involved in RBC alloimmunization [99]. MZ B cells and MZMs can receive and integrate signals from the local microenvironment that are likely important in dictating the overall immune response following RBC alloantigen exposure [72 100 101 102 103 104 105 106 107 108].

Fig. 1. Red blood cells interact with distinct immune constituents following transfusion.

Studies utilizing various preclinical models indicate that transfused red blood cells (RBCs) can engage specific immune cells in the spleen, such as marginal zone (MZ) B cells, macrophages in the marginal zone, and bridging channel dendritic cells (DCs). These interactions can initiate several unique immune pathways potentially leading to the production of alloantibodies. Nonetheless, transfusion of allogeneic RBCs does not invariably lead to alloimmunization. In some instances, RBC transfusions may promote antigen-specific non-responsiveness, likely by modifying the response capabilities of alloantigen-specific immune cells.

The ability of RBC transfusions to induce a state of antigen-specific non-responsiveness has been an intriguing observation from preclinical studies. This phenomenon appears to be influenced, at least in part, by the density of the RBC alloantigen on the cell surface. Unlike major histocompatibility complex (MHC) antigens that are highly polymorphic, yet utilize similar features to present peptides to T cell receptors (TCRs) [113], RBC alloantigens exhibit remarkable diversity in terms of density, composition, structure, and lateral mobility on the RBC membrane. These differences not only can impact the outcome of incompatible transfusion [114], but are also likely to impact the nature of B cell receptor (BCR) engagement and subsequent signaling events following alloantigen exposure. Unlike most receptor-ligand interactions, the BCR must respond to a vast array of antigenic determinants without prior exposure, a unique challenge that may render it sensitive to variations in antigen density, orientation, and overall presentation [115 116]. Low antigen densities on RBCs may result in suboptimal BCR clustering and signaling, potentially leading to B cell anergy or deletion. Conversely, high antigen densities could drive excessive BCR crosslinking, causing hyperstimulation and apoptosis ( Fig. 2 ). The presence of pre-existing alloantibodies or complement deposition on the RBC surface can further modulate the outcome of BCR engagement, potentially converting a tolerogenic signal into an immunogenic one [78 86 117 118 119 120 121 122 123]. Collectively, these findings suggest that the density and configuration of RBC alloantigens, coupled with factors like antibody opsonization and complement activation, can fine-tune the strength of BCR signaling, dictating whether tolerance or immunity ensues. Uncovering these pathways could facilitate the development of strategies to modulate alloimmunization risk or conversely, induce antigen-specific tolerance for therapeutic applications.

Fig. 2. Different antigen densities on red blood cells (RBCs) may influence host immune outcomes following transfusion.

Unlike human leukocyte antigens, which exhibit considerable sequence variability but maintain similar structure and function, RBC alloantigens often represent distinct macromolecular structures on the RBC surface. These structural differences can result in varying levels of surface expression, lateral mobility within the plasma membrane, and the arrangement of allogeneic epitopes perceived by a transfused recipient. While further studies are necessary, variations in antigen density may directly influence B cell responses, potentially inducing anergy, apoptosis, or alloimmune responses. Understanding the key factors that determine non-responsiveness versus alloimmunity is crucial for the successful use of RBCs in efforts to deliberately induce antigen-specific tolerance.

2.1. Harnessing the potential of RBCs as immunomodulatory agents

Unlike many cells, RBCs lack the ability to divide (post-mitotic) after maturation, allowing surface antigens to persist without being replaced. This unique property enables RBCs to serve as a stable platform for displaying exogenous antigens on an otherwise “self” surface, potentially facilitating antigen presentation to the host’s immune system without triggering innate immune activation typically caused by exposure to common features of microbes [124 125] ( Fig. 3 ). Since RBCs do not generate new membrane structures post-maturation, introduced membrane modifications in the form of tethering antigens to the RBC surface can be preserved throughout the lifespan of the cell. This approach has been exploited to track RBC survival after transfusion using cell surface biotinylation and various antigen structures have likewise been coupled using similar chemical approaches at different densities on the cell surface [122 123 126 127 128]. Conjugating antibodies of known specificity, aptamers, or other strategies to link antigens to the cell surface have also been proposed [129 130]. These approaches also allow antigens to be attached to autologous RBCs, reducing alloantigen exposure that may occur following allogeneic RBC exposure. However, the attachment sites for antigens or similar linkage chemistries are difficult to control, potentially creating neo-antigenic determinants where chemical coupling occurs that may result in distinct epitopes from the target antigen. Strategies that introduce specific chemical tags on the antigen itself can reduce variability in epitope formation on the antigen itself and may preserve a common antigen orientation but do not eliminate heterogeneity in attachment sites on the RBC surface. As a result, while chemically modifying the RBC surface provides flexibility for introducing distinct antigenic determinants, the lack of control over the specific attachment sites may limit this approach’s overall utility, given possible heterogeneous antigen orientations and unintended neo-epitope formation that could occur. However, additional studies are certainly needed to define the extent to which these theoretical challenges may limit this approach.

Fig. 3. Red blood cells (RBCs) provide an attractive chassis to build vehicles of immunological tolerance.

Unlike nucleated cells that readily divide and possess the capacity to transcriptionally respond to environmental stimuli, RBCs lack much of the cellular machinery required for protein synthesis and membrane modifications. Consequently, modifications to an RBC surface may be sustained for the cell’s entire lifespan. Given these unique properties, RBCs can serve as a unique host-derived scaffold upon which chemical and enzymatic modifications can be introduced, endowing these cells with distinct properties that may allow them to act as intentional vehicles of antigen-specific tolerance. Similarly, generating RBCs from induced pluripotent stem cells (iPSCs) could provide a genetically engineered and reproducible source of RBCs with unique properties that could be used beyond their oxygen-carrying capacity to modulate recipient immunity. Additional studies involving transfusion of RBCs loaded with antigens intracellularly to modulate recipient immune function are not shown.

Recent studies have explored innovative strategies to more precisely modify RBC surface antigens like the KEL antigen for controlled presentation of exogenous antigenic cargo. One promising approach to accomplish this involves genetically engineering a “sortase-tagable” modification on the KEL antigen [131]. This allows distinct antigens of interest to be covalently attached to KEL on RBC membranes using sortase enzymes in a predictable manner. In this way, the density, orientation, and localization of the attached antigen cargo would more predictably mimic that of the native KEL antigen. By leveraging the known distribution pattern of an endogenous RBC surface protein, the issues associated with non-specific chemical cross-linking methods can potentially be circumvented. Indeed, proof-of-concept studies have demonstrated successful attachment of model antigens to engineered KEL on RBCs, and transfusion of these antigen-displaying RBCs was able to modulate immune responses against the coupled antigen in vivo [131]. However, realizing such an approach clinically would require genetic modification of hematopoietic stem cells to produce RBCs constitutively expressing the sortase-tagable KEL variant. To avoid this limitation, recent approaches have employed a chemoenzymatic approach using a peptide that specifically engages glycophorin A (GPA) to load cargo on RBCs independent of genetic RBC engineering [132]. Preclinical data demonstrate that transfusion of murine RBCs expressing GPA in the absence of inflammation can induce antigen specific tolerance [76], suggesting that this approach may be useful in strategies designed to induce antigen specific immune non-responsiveness. Indeed, this strategy reduces antibody formation against a distinct cargo attached to GPA on the RBC surface [132]. While this strategy shows promise for controlled antigen display, other considerations like potential neo-epitope formation at the site of attachment, effects on RBC survival, and scalable manufacturing would need to be addressed. However, these approaches represent innovative applications that may overcome the limitations of more commonly employed RBC surface modification techniques.

An emerging and promising approach to leveraging RBCs as a chassis to present antigen cargo in a potentially tolerogenic fashion involves directly engineering RBCs to display distinct antigen configurations on their surface. This strategy circumvents the need for chemical modification or the attachment of proteins through various deliberate tagging approaches. While a variety of approaches have emerged, the use of induced pluripotent stem cell (iPSC) differentiation protocols to generate mature RBCs from genetically engineered precursor cells appears to be a promising strategy [133 134 135]. By introducing genetic modifications in the iPSCs, the resulting RBCs can constitutively express chimeric antigen constructs fused to RBC membrane proteins. This same strategy can be used to induce ectopic expression of a protein of interest or even express a native RBC alloantigen at different densities that may be more likely to induce tolerogenic outcomes. A major advantage of this genetic engineering approach is the ability to precisely control the type of antigen displayed, its density, and its localization pattern on the RBC surface. However, one potential challenge is that genetically engineered cells may expose recipients to other alloantigens present on the RBC surface depending on the genetics of the original donor used to derive the iPSCs and the blood type of the recipient. Efforts are being undertaken to reduce the likelihood that this will occur by eliminating or modifying the most common blood group targets, but the impact of these approaches on RBC survival and other RBC characteristics remains relatively unexplored [133 134 135].

While currently not feasible for routine RBC transfusion due to high production costs, generating an antigen-engineered RBC product capable of inducing antigen-specific tolerance would likely justify the associated expenses. Such a product could potentially treat autoimmune disorders, allergies or prevent immune responses against protein therapeutics, without compromising systemic immunity. Similar strategies could possibly be employed to eliminate or reduce alloimmunity following transfusion or transplantation [136 137]. However, translating this approach clinically requires overcoming challenges that include scalable manufacturing of genetically modified RBCs and ensuring product safety and reproducibility. The efficacy of RBC-based approaches in patients who have already developed alloantibodies also remains uncertain. This is particularly relevant given that several studies have shown passively administered antibodies can either inhibit or augment RBC-induced alloimmunization [78 86 121], potentially converting a normally tolerogenic signal into an immune response. While strategies to reduce alloantibody levels before initiating RBC tolerance therapies could be employed, completely negating the impact of residual alloantibodies on such therapies may prove challenging. Further research is needed to determine if and how RBC-based tolerance induction strategies can be effectively implemented in patients with pre-existing alloantibodies. Nonetheless, the ability to generate a highly defined and reproducible antigen-displaying RBC population represents an intriguing strategy for the possibility of controlled induction of antigen-specific tolerance.

While many studies have examined the ability of RBCs as possible vehicles to prevent antibody formation through the possible manipulation of antigen specific B cells, several studies have also used RBCs to encapsulate antigens as a novel approach to induce antigen-specific T cell tolerance. This approach leverages strategies employed to use RBCs as drug carriers in general and has the potential to induce T cell anergy or apoptosis by presenting antigens to T cells in the absence of inflammation [138]. Several techniques have been explored for intracellular antigen loading into RBCs, including hypotonic dilution, electroporation, and using transient pore-forming agents. Alternatively, genetic engineering of RBC precursors can enable endogenous expression of therapeutic proteins within RBCs. Encapsulated antigens are protected from degradation and can be gradually released over the RBC lifespan [139], potentially inducing antigen-specific tolerance through engulfment of RBCs expressing antigens and presentation to antigen specific T cells in the absence of inflammation, thereby possibly inducing antigen specific T cell anergy or apoptosis.

It should be noted that in contrast to inducing tolerance, RBCs can also be engineered to enhance desirable immune responses as an alternative form of immunotherapy. As noted previously, enhancing a recipient’s anti-tumor immunity has become a cornerstone for advancing new cancer treatment strategies. Leveraging RBCs to boost anti-tumor immunity has likewise been achieved. This has been accomplished by loading RBCs with distinct antigens and immune activators in an effort to stimulate desirable immune responses aimed at enhancing cancer treatment [140 141]. Key features of RBCs, including modification strategies, that result in tolerant versus immunostimulatory outcomes need to be fully defined.

3. Summary

RBC transfusion represents the oldest and most widely used form of cellular therapy. While traditionally employed to augment oxygen carrying capacity in the setting of anemia, the unique biological properties of RBCs may render them as attractive vehicles for delivering antigenic cargo to induce antigen-specific tolerance. Preclinical studies have demonstrated the promising potential of RBC transfusion to induce a state of immune non-responsiveness against specific antigens. However, to fully harness this approach, further investigations are warranted to elucidate the mechanistic underpinnings by which transfusion-induced tolerance occurs and understanding key levers that dictate tolerance or increased immunity outcomes. Delineating these pathways could guide optimization strategies for engineering RBC products tailored to reliably elicit the desired clinical endpoint. The relative efficacy of different antigen-decoration methodologies in promoting antigen-specific tolerance induction also merits additional evaluation. Notwithstanding the need for additional research, current evidence suggests that RBCs possess inherent attributes that position them as relatively simple yet potentially effective platforms for inducing antigen-specific immunotherapy. By leveraging their unique traits, RBC-based therapies could provide an innovative addition to a long-standing clinical intervention, repurposing transfusion for targeted immune modulation.