Immunological platelet transfusion refractoriness: current insights from mechanisms to therapeutics

Abstract

Although there have been tremendous improvements in the production and storage of platelets, platelet transfusion refractoriness (PTR) remains a serious clinical issue that may lead to various severe adverse events. The burden of supplying platelets is worsened by rising market demand and limited donor pools of compatible platelets. Antibodies against platelet antigens are known to activate platelets through FcγR-dependent or complement-activated channels, thereby rapidly eliminating foreign platelets. Recently, other mechanisms of platelet clearance have been reported. The current treatment strategy for PTR is to select appropriate and compatible platelets; however, this necessitates a sizable donor pool and technical assistance for costly testing. Consolidation of these mechanisms should be of critical significance in providing insight to establish novel therapeutics to target immunological platelet refractoriness. Therefore, the purposes of this review were to explore the modulation of the immune system over the activation and elimination of allogeneic platelets and to summarize the development of alternative approaches for treating and avoiding alloimmunization to human leukocyte antigen or human platelet antigen in PTR.

Plain Language Summary

Platelet transfusion is a critical treatment for patients with a severely reduced platelet count and significant bleeding symptoms. However, some patients do not respond to transfused platelets, especially those with repeated transfusions and malignant hematologic disorders, which may increase the burden of disease. In this review article, the authors outline how immunological factors contribute to the failure of platelet transfusions and conventional therapies. Although antibody-mediated platelet removal is often considered the predominant immunological mechanism, studies have shown that CD8⁺ T cells also play a unique role in platelet clearance. The authors also cover the prospects and challenges of alternative treatment strategies in clinical practice.

Keywords:

• Antibodies
• CD8+ T cells
• HLA
• HPA
• platelet transfusion refractoriness
• therapeutics

Introduction

Platelets are a scarce resource, given that they are solely obtained through donations. When the platelet count falls below a predetermined threshold, prophylactic, or therapeutic platelet transfusion is an indispensable clinical practice. However, some patients, particularly those requiring ongoing platelet transfusion and with hematologic disorders, may exhibit refractoriness to platelet transfusion. Platelet transfusion refractoriness (PTR) is a lasting, unsatisfactory post-transfusion platelet count increment after two or more consecutive transfusions of adequate doses of ABO-matched platelets.¹ PTR may induce a series of adverse events, such as spontaneous or life-threatening bleeding, prolonged hospital stay, and increased medical costs.^2–4 It is an urgent clinically malignant event that worsens the patient’s condition and limits blood resource availability. Therefore, this review provides a systematic overview and commentary on the clinical status, mechanisms, and treatment of PTR. It highlights the regulatory role of the immune system in the activation and clearance of allogeneic platelets and outlines the progress to date in alternative approaches to treat and prevent human leukocyte antigen (HLA) alloimmunization in PTR.

Definition and clinical situation of platelet transfusion refractoriness

A variety of metrics are used to assess the efficiency of platelet transfusion, and the corrected count increment (CCI) is frequently employed to diagnose PTR.⁵ The calculation formula of the CCI is the post-transfusion platelet count increment multiplied by the body surface area (m²) of the patient, divided by the product of the number of platelet transfusions and 10¹¹. The Trial to Reduce Alloimmunization to Platelet (TRAP) study defined PTR as a 1-h CCI value of less than 5000 after two consecutive platelet transfusions.¹ The inclusion of the patient’s body surface area and the amount of platelet transfusion can effectively minimize individual differences and lead to a more accurate assessment of the effectiveness of platelet transfusion. Specific platelet transfusion volumes are not routinely provided. When the body surface area and amount of platelet transfusion are unknown, the post-transfusion platelet count can be used as a simple index for clinical practice. Therefore, clinicians use platelet counts in routine healthcare, whereas CCIs are reserved for research. The administration of leukocyte reduction and irradiated platelets has significantly reduced the frequency of transfusion-related adverse events.¹ Although the universal application of leuko-depleted platelets has dramatically reduced the incidence of alloimmunization and PTR, data from the TRAP group, which enrolled 530 patients, showed that 17–21% of those patients still developed alloantibodies, 3–4% of whom became refractory to platelet transfusion due to alloimmunization.¹ Clearly, PTR remains a challenging clinical issue that requires attention.

The causes of PTR can be divided into two categories, namely nonimmune and immune factors. Nonimmune factors include infection, fever, diffuse intravascular coagulation, splenomegaly, and medications.⁶ However, these factors are not always easy to identify. In practice, nonimmune refractoriness is considered when no anti-platelet antibodies are identified, making it a diagnosis of exclusion. Although nonimmune factors cause most PTR cases, immune factors still account for approximately 20%.^1,7 Furthermore, another large trial performed by the TRAP group demonstrated that lymphocytotoxic antibody positivity ranked as the most severe among all adverse factors affecting PTR.⁸ It is well known that immune responses can be divided into innate and adaptive, with the latter further subdivided into humoral and cellular immunity. Humoral immunity is widely considered the sole cause of immune-driven PTR. Platelet antibodies that cause PTR include HLA, human platelet antigen (HPA), ABO, and CD36 antibodies.⁹ However, the role of cellular immunity in PTR is frequently disregarded. Additionally, how antiplatelet antibodies function at the cellular and molecular levels remains unclear.

The present approach for treating PTR involves the selection of suitable platelets. Random donor platelets are the first to be routinely used clinically for transfusion. When post-transfusion platelet counts fail to meet expectations and subsequently the patient is diagnosed with HLA alloimmunization-mediated PTR, three platelet types are considered, including HLA-matched, HLA antigen-negative that circumvent HLA antibodies, and cross-matched platelets.¹⁰ There is no definitive conclusion regarding which of these three options is most suitable for HLA antibody-mediated PTR. Owing to the polymorphism of HLA molecules and the increasing demand for platelets, the current resources of the HLA-matched platelet donor pool are limited, resulting in a lack of timely supply of platelets, which is especially problematic for patients who require regulation transfusion, such as during cytostatic therapy for malignant hematologic disorders. Therefore, researchers have attempted to explore alternative therapeutic approaches based on established mechanisms of platelet clearance.

Mechanisms of immunological platelet transfusion refractoriness

Antibodies against HLA, HPA, and CD36 molecules on the platelet surface can induce the clearance of foreign platelets.^11–15 Genetic polymorphisms in these molecules contribute to the development of antibodies.^16–18 Additionally, the role of CD8⁺ T cells in platelet destruction has been reported.^19–22 Here, we summarize these potential mechanisms (Figure 1).

Figure 1. Mechanisms of platelet clearance. Different types of antiplatelet antibodies bind to the corresponding antigens on the platelet surface (including HLA, HPA, and CD36 antigens), all of which can activate platelets through an FcγR-dependent or complement-activated channel, leading to the rapid removal of foreign platelets in the spleen. Desialylation is an additional mechanism of platelet destruction to be specific, anti-GPIbα antibodies induce neuraminidase-1 (neu-1) transfer to the platelet surface, causing platelet desialylation. Desialylated platelets are destroyed primarily in the liver via the ashwell morell receptor. Simultaneously, platelet activation and desialylation form a positive feedback loop. Compared to anti-GPIbα antibodies, anti-GPIIb/IIIa antibodies initiate platelet desialylation via the FcγRIIa-dependent pathway and, to a lesser extent, platelet activation. CD8+T cells can also induce Neu1 expression and platelet desialylation. HLA, human leukocyte antigen; HPA, human platelet antigen; GP, glycoprotein.

HLA-mediated platelet transfusion refractoriness

Platelets are small, anucleate blood cells. In contrast to erythrocytes, platelets possess a substantial quantity of HLA molecules, comprising both native and acquired sources.²³ Platelets only express HLA class I molecules generally; however, several studies have reported that platelets can transiently express HLA class II molecules in the setting of immune diseases^24,25 but not under normal physiological conditions. When platelets are transfused into a recipient, the recipient becomes exposed to allogeneic donor HLA molecules on the platelet surface. Multiple transfusions²⁶ and pregnancies²⁷ increase allogeneic antigen exposure, potentially leading to alloimmunization. Recipient antigen-presenting cells (APC) process and present these allogeneic HLA antigens to the T-cell receptors of recipient T-helper cells. The above process is similar to the normal immune response, generally called the indirect pathway.²⁸ Additionally, the activation of inducible nitric oxide synthase within recipient APC plays a substantial role in platelet antigen processing within this pathway,²⁹ which may be an effective precaution for the production of alloantibodies. Furthermore, different processing pathways, such as endosomal and non-endosomal pathways in recipient APC, can affect IgG subclass formation.³⁰ Interestingly, the CD40 ligand (CD40L), a pivotal signaling molecule for T cell-dependent activation of B cells, can be expressed by platelets.³¹ Both platelets and platelet-derived membrane vesicles can transmit activation signals to B cells via self-expressed CD40L, ultimately inducing IgG antibody production.^32,33

Once a patient generates HLA alloantibodies, several clinical side effects and unsatisfactory platelet count increments in a subsequent platelet transfusion may occur. HLA antibodies can induce platelet activation and phagocytosis by macrophages, leading to platelet clearance.^11,12 Rijkers et al.¹¹ confirmed that FcγRIIa, the platelet Fc receptor, plays an important role in the above processes. They showed that certain HLA antibodies can cross-link HLA antigens with FcγRIIa in an intra-platelet-dependent way to activate platelets. Apart from HLA antibodies, a platelet activation role for other platelet-specific antibodies, including heparin-induced thrombocytopenia (HIT)-related antibodies,³⁴ anti-FcγRIIa antibody IV.3,³⁵ and platelet-specific antibodies targeting platelet membrane proteins, such as CD9³⁶ and GPIIb/IIIa,³⁷ has been reported in a FcγRIIa-dependent pathway. FcγRIIa is a single-chain transmembrane glycoprotein with an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic tail and two extracellular Ig-like domains.^38,39 The second Ig-like domain of FcγRIIa, which mediates the binding to IgG,⁴⁰ contains two common alleles at amino acid 131 that produce two variants with different responsiveness, namely histidine (H) and arginine (R).⁴¹ The polymorphism of H/R influences the binding affinity of different IgG subclasses for FcγRIIa.⁴² The role of FcγR-expressing mononuclear macrophages in mediating the phagocytosis of antibody-coated platelets is widely recognized. This phagocytosis occurs primarily but not exclusively in the spleen, which may be related to the slow passage of platelets through the splenic sinus with high FcγR expression on macrophages.^43,44 HLA antibodies are also capable of inducing complement activation on platelets utilizing the classical pathway, leading to the formation of a membrane attack complex and platelet activation followed by platelet clearance.¹² Increased galactosylation, and to a lesser extent, increased sialylation, can enhance complement activation, and galactosylation has a greater enhancement¹⁵; this is also observed with HPA antibodies.¹⁵

The presence of alloantibodies does not equate to the development of PTR. Data from the TRAP study showed that a significant number of patients generated alloantibodies after transfusion but did not become refractory.¹ Beligaswatte et al.⁴⁵ suggested that the amount of antibody binding, which is expressed as the mean fluorescence intensity, is correlated to the development of PTR. However, the data from the Rijkers et al.¹¹ study did not support the above relationship. This discrepancy indicates that the immunological PTR mechanism is complex and potentially multifactorial. We hypothesize potential mechanisms behind IgG antiplatelet immunity in platelet refractoriness. First, Seielstad et al.¹⁶ proposed a role for single nucleotide polymorphisms (SNPs) of the neurexophilin 2 gene in HLA alloimmunization. A small allele variant of the neurexophilin 2 gene seems to increase the risk of HLA class I antibody generation in previously pregnant women.¹⁶ It may be worth exploring whether genetic variation takes part in HLA alloimmunization in the context of platelet transfusion. Second, only partial HLA antibodies enhance platelet clearance by cross-linking FcγRIIa. We speculate that this may be influenced by the binding affinity of the IgG antibody for FcγRIIa, varying among IgG subclasses.⁴⁶ Moreover, the polymorphism of the gene encoding human FcγRIIa at position 131 has been demonstrated to influence the affinity of IgG2 for FcγRIIa.⁴² This finding also implies that larger-scale genetic research regarding which variants are associated with platelet-derived alloimmunization is needed. Third, patients have highly varied levels of IgG-Fc glycosylation patterns, which may cause different levels of complement activation and clearance.¹⁵ Finally, HLA expression on platelets is highly variable among different populations, and different HLA antibody combinations have markedly different forces on platelets.^12,47 Therefore, it is plausible that only some patients carrying the antibodies develop PTR when randomized platelets are routinely transfused.

Most of the literature concerning immune-mediated PTR is focused on antibody interactions with antigens distributed on the platelet surface. Negative results of platelet antibody detection techniques invariably allow immune-mediated PTR to be disregarded. In this instance, PTR is often attributed to nonimmune factors, though sometimes specific nonimmune factors cannot be identified. In 2004, data from murine models demonstrated that, in the absence of CD8⁺ T cells, the IgG antiplatelet immune response is enhanced, as evidenced by earlier antibody production and higher total antibody levels. Further analysis of antibody isotypes revealed that IgG1 production was remarkably inhibited, while IgG2a production was promoted,¹⁹ potentially suggesting an inhibition of the antibody response against allogeneic platelets by CD8⁺ T cells. Furthermore, CD8⁺ T cells appeared to negatively regulate IFN-γ secretion by natural killer cells in this study.¹⁹ In 2016, Arthur et al.²⁰ used B-cell-deficient mice to demonstrate considerable platelet clearance in immunized recipients by the major histocompatibility complex (MHC) following MHC-mismatched platelet transfusion, whereas no significant clearance was observed in nonimmunized recipients. Simultaneously, before transfusing MHC-mismatched platelets, CD8⁺ T cells were eliminated in another group of immunized recipients, and in contrast to the isotype control, platelet clearance was significantly weakened in this group.²⁰ Thus, these authors deduced that CD8⁺ T cells also play a role in immune-mediated platelet clearance in the absence of alloantibodies targeted to platelets.²⁰ These two studies confirmed that CD8⁺ T cells are involved in the clearance of foreign platelets and may break the limited perception of whether PTR is mediated by immune factors based on antibody detection. There are complex interactions between CD8⁺ T cells and alloantibodies. Further novel investigations are needed to explore the relationship between CD8⁺ T cells and antibodies as well as the process by which CD8⁺ T cells eliminate allogeneic platelets.

HPA-mediated platelet transfusion refractoriness

Antibodies against HLA are the primary elicitors of immune-mediated PTR. If HLA antibodies cannot be detected, other uncommon antibodies should be suspected, such as antibodies to HPA and CD36. Various HPA antigens are distributed on platelet glycoproteins (GPs). Twelve HPA molecules are divided into high-frequency antigens named “a” and low-frequency antigens named “b” within each of their six biallelic groups, including HPA—1, −2, −3, −4, −5, and − 15. These six HPA antigens of high frequency are reportedly linked to PTR since they may trigger alloantibody development under conditions of pregnancy, multiple-platelet transfusions, and transplantation.⁹ There are multiple reports on the genetic polymorphisms of HPAs in various areas, which have demonstrated substantial distinctions in the allele and genotype frequencies of HPAs between countries, regions, and races.^17,18 Recognizing regional variations in HPA polymorphisms may help predict and avoid the risk of alloimmunization.

Similar to HLA antibodies, HPA antibodies can activate platelets through an FcγR-dependent or complement-activated channel, leading to the rapid removal of foreign platelets.^{13–15,36,37} In addition to alloantibody-mediated clearance, platelets are also cleared by autoantibodies. In immune thrombocytopenia (ITP), it has become evident that platelets can be removed via desialylation.^37,48 Platelet desialylation is a process in which neuraminidase-1 (Neu-1) induces the removal of terminal sialic acids predominantly derived from GPs on the platelet surface, thereby allowing the penultimate β-galactose residues to be exposed. The desialylated platelets can be recognized by the Ashwell Morell receptor, which is primarily expressed on hepatocytes and then phagocytosed.²¹ Anti-GPIbα antibodies can induce platelet activation and desialylation. These two events strengthen each other, forming a positive feedback loop.⁴⁸ Recently, it was shown that anti-GPIIb/IIIa antibodies are responsible for platelet desialylation by FcγRIIa-dependent mechanisms, which differs from anti-GPIbα antibody-mediated platelet desialylation by FcγR-independent mechanisms mainly occurring in the liver.^37,49 Whether one or more HPA alloantibodies on GPs can also trigger platelet clearance by desialylation warrants further study. Furthermore, CD8⁺ T cells can lead to Neu-1 expression on platelets and platelet desialylation, resulting in platelet clearance in ITP.^21,22 The role of CD8⁺ T cells in platelet clearance via platelet desialylation in patients with PTR has not been studied. Recognizing the mechanism of cellular immunity in PTR may lead to innovative therapeutic interventions in the future.

CD36-mediated platelet transfusion refractoriness

Different proportions of distinct ethnic populations have no or limited GPIV/CD36 expression, which predisposes to the generation of anti-CD36 antibodies after alloimmunization. Single-nucleotide variations are a substantial contributor to differential CD36 expression.⁵⁰ These genetic mutations seem to vary within diverse ethnic populations.^51,52 Additionally, several molecular regulations of CD36, such as alternative splicing products, also play an essential role in CD36 expression. This explains why some people are CD36 expression-deficient without any potential coding mutations.⁵³

Notably, donors’ platelets are mainly tested for HLA rather than HPA or CD36. Although the percentage of PTR caused by anti-HPA or anti-CD36 antibodies alone is low, reasonable suspicion and timely diagnosis of such clinical conditions will potentially help resolve thrombocytopenia, which is important for patients, and reduce wastage of blood resources.

Treatment of platelet transfusion refractoriness

Currently, selecting suitable platelets is key to improving immunological platelet refractoriness, and several therapeutic regimens are available. The first protocol is to choose cross-matched platelets. This approach requires cross-reactivity between the patient’s serum and the donor’s platelet without testing donor platelet antigen or patient platelet antibody type. It is simple, quick, and compatible with all types of platelet antibodies. However, there are not enough platelets available to make compatibility tests for hypersensitive patients. Besides, the transfusion of cross-matched platelets is thought to increase the risk of additional alloimmunization. As a result, re-testing for potential cross-matches is generally recommended.

The second protocol involves selecting HLA-matched platelets, which requires HLA-type testing for both donor and recipient platelets. The degree of HLA match is graded according to the A and B loci of HLA and can be divided into five grades from high to low as follows: A, BU, BX, C, and D.¹⁰ Higher matching grade platelets, such as grade A and BU, can provide positive transfusion outcomes, while lower matching grade platelets such as grade BX, C, and D cannot and can only be compared to the outcomes of random platelets. However, finding HLA-matched platelets is challenging, particularly for patients with uncommon HLA antigens, necessitating an incredibly sizable donor platelet HLA gene pool. Among Dutch blood donors, the most prevalent haplotypes of the HLA-B antigens were found to be HLA-B7, -B12, -B8, and -B35.⁵⁴ Subsequently, Saris et al.⁴⁷ found that HLA-B8, -B12, and -B35 had low expression level on platelets of part of the population, and these platelets with low HLA-B expression induced a smaller degree of antibody-mediated platelet opsonization than platelets with high expression in vitro. Theoretically, the HLA-B matching standards for such donors could be lowered, which would facilitate the establishment of a larger platelet donor pool. Further clinical trials are needed to confirm this suspicion. Anti-HLA-C antibodies have demonstrated an involvement in PTR,⁵⁵ and thus, HLA-C should also be considered in the classification system for matching degrees.

The third protocol involves choosing the corresponding antigen-negative platelets by measuring the anti-platelet antibodies in the patient. This approach expands the pool of available donor platelets compared to HLA-matched platelets, but also increases the risk of further alloimmunization. For patients with PTR who have merely anti-HPA antibodies, the corresponding HPA-negative platelets can be selected.⁵⁶

The aforementioned schemes are matched at the antigen level; however, a new matching strategy based on HLA epitopes has been proposed. Epitopes are structures comprising several amino acid residues in a linear or discontinuous manner, which can be divided into private epitopes, specific to a single antigen molecule, and public epitopes, which multiple antigens can share.⁵⁷ Duquesnoy et al.⁵⁸ designed HLAMatchmaker software that can evaluate HLA compatibility based on HLA epitopes. A double-blind, randomized prospective trial demonstrated that HLA-epitope-matched platelets identified using HLAMatchmaker were not inferior to traditional HLA-matched platelets regarding transfusion effectiveness.⁵⁷ In addition, the epitope score was shown to be linked to the proportion of successful HLA-mismatched transfusions.⁵⁹ The limitation of this approach is that it demands the support of HLA high-resolution technology, requiring additional time and economic costs. A single-center trial showed equivalent efficacy of HLA-mismatched platelet transfusions compared to HLA-matched platelet transfusions in PTR patients without donor-specific antibodies (DSAs).⁵⁹ Still, HLA-mismatched transfusion with less than three DSAs are often successful.⁵⁹ When HLA-matched platelets are unavailable, considering both DSA and epitope score to find donor platelets can help minimize additional immunological risk.

Prospects for the therapy of platelet transfusion refractoriness

Based on the known mechanism of interaction between HLA antibodies and platelets, various approaches targeting HLA antigens, HLA antibodies themselves, or antibody-mediated signaling pathways have been designed to mitigate platelet activation and clearance.

Targeting the HLA antigens in donor platelets

Reduction or even elimination of HLA antigens on donor platelet surfaces to reduce the immunogenicity of platelets while preserving their normal physiological function has been a topic of interest for the prophylaxis and treatment of HLA alloimmunization and refractoriness. The concept of using platelets stripped of HLA antigens by an acid solution to treat PTR was first proposed in 1984.⁶⁰ In 1991, Shanwell et al.⁶¹ presented a successful case of a patient with anti-HLA antibodies-mediated PTR whose CCI significantly improved after platelet transfusion treated with acid (pH 2.8 at 0°C). The study also indicated that the viability of acid-treated autologous platelets transfused back into healthy individuals was moderate.⁶¹ The following year, another group⁶² used the same acid treatment method described by Shanwell et al.⁶¹ and infused acid-treated platelets into one patient; however, no significant therapeutic effect was obtained. With slight adjustments to the conditions of acid treatment, such as pH and temperature, a few patients achieved great transfusion results without adverse effects.^63,64 Although successful cases are rare, the findings indicate the potential feasibility of using HLA-stripped platelets. Nevertheless, it should be noted that the function of platelets after acid treatment may be compromised,^65,66 and these platelets retain a portion of HLA antigens, which could potentially lead to immunogenicity. Recently, with the optimization of acid treatment technology, the platelet proteome,⁶⁷ viability, and function^68,69 have largely remained. These outcomes create the opportunity for a clinical trial, and the function and immunogenicity of acid-treated platelets require further validation in vivo.

Platelets have always been in limited supply, and those obtained through donation will probably be unable to meet increasing clinical demands each year. With rapid advances in biological and gene editing technology, HLA-universal platelets, which refer to blood components with reduced or even no HLA antigen expression and that can be supplied continuously, have been successfully produced in vitro.^70,71 It is an appealing prospect for transfusion medicine that will not only alleviate the resource deficit but also prevent HLA alloimmunization and refractoriness. Several starting cells including CD34⁺ hematopoietic progenitor cells,⁷² pluripotent stem cells (PSCs), induced human pluripotent stem cells (iPSCs),^70,73 and embryonic stem cells (ESCs)⁷⁴ have been extensively studied as an ex vivo source of platelet manufacture. The disruption of beta-2 microglobulin, a light chain constituent pivotal for the synthesis and expression of HLA molecules, forms the basis of HLA depletion on platelet surfaces.⁷⁵ Various gene editing technologies have attempted to produce HLA-universal platelets, yielding varying results artificially. For instance, RNA interference techniques⁷¹ can lower HLA expression, whereas TALENs⁷⁰ or the CRISPR/Cas9 system^76,77 can completely eradicate HLA expression. The clinical utilization of these artificial platelets is constrained by their ability to adhere to therapeutic criteria concerning quality, quantity, and safety.⁷⁸ Until the work of the Eto group,⁷⁹ platelets had not been produced at the level required for one apheresis transfusion unit. The group found that, in addition to shear stress, a known regulator important for platelet shedding,⁸⁰ turbulence is another key physical factor in platelet release. The Eto group then produced 100 billion functional platelets using a turbulent flow-based bioreactor, VerMES. The functionality of artificial platelets has been validated in animal models.^76,79 Notably, the first clinical trial in which platelets cultured from autologous iPSCs were successfully transfused into a patient with severe aplastic anemia and refractoriness to platelet transfusion due to anti-HPA antibodies occurred in 2022.⁸¹ This finding indicates the potential application of HLA-universal platelets derived from iPSCs as a renewable and low-immunogenicity alternative resource for platelet transfusion. However, the current culture procedure for artificial platelets is complicated and time-consuming, and large-scale manufacture has not yet been achieved, making its cost much higher than that of donor platelets. Although human PSCs are an attractive and renewable resource, they can lead to a wide variety of genetic changes in culture.⁸² For example, dominant negative mutations in TP53, the most commonly mutated gene in human cancer, occur during prolonged culture of ESCs.⁸³ Moreover, the application of gene editing techniques can lead to off-target effects, consequently producing unexpected genomic changes and even increasing cancer risk.^84,85 Thus, vigilance to the possible risks associated with these genetic instabilities is needed. Supported by studies aiming to understand the mechanisms of genome editing techniques and sources of off-targeting, research efforts have been made to optimize strategies to mitigate off-target effects and ensure safety in clinical applications.^86–88 Overall, HLA universal platelets have great potential as an alternative treatment option to transfusion.

Targeting the response of HLA antibodies to antigens

Several studies have revealed that HLA antibodies can activate platelets and facilitate the uptake of platelets by macrophages through the FcγRIIa-dependent and complement activation pathways. Inhibition of these two pathways can reduce the removal of platelets. Studies suggest that C1 esterase inhibitors, aiming to complement activation through the classical pathway, and spleen tyrosine kinase (Syk) inhibitors IV.3 can, in a dose-dependent manner, block platelet activation in vitro and cause the inhibitory effect to no longer increase when the concentration reaches a certain threshold.^11,12 HMPL-523, a novel Syk inhibitor, has been shown to be safe and effective in patients with ITP in a randomized, double-blind phase 1b clinical study,⁸⁹ alluding to the role of Syk inhibitors in PTR.

A pilot clinical trial, comprising 10 patients who were diagnosed with HLA antibody-mediated PTR, was conducted between 2015 and 2017.⁹⁰ Eculizumab, a C5 complement inhibitor, can obstruct three pathways of complement activation and was used to evaluate the treatment effectiveness of immune-driven PTR. It was shown that platelet CCI dramatically improved following eculizumab treatment in 4 of 10 patients, both with HLA-compatible and HLA-incompatible platelet transfusions.⁹⁰ However, it is difficult to determine the features of the treatment-responsive group and precisely calculate the rate of drug effectiveness due to the limited sample size. Nonetheless, eculizumab is too expensive to be used universally.

Targeting the anti-HLA antibodies in recipients

Webber et al.⁹¹ proposed the concept of HLA-Fc fusion proteins that can neutralize cognate antibodies and even selectively deplete antibody-producing cells, inhibiting the initiation of humoral immunity. The HLA-Fc fusion protein is composed of the extracellular domains of class I HLA, which can bind to surface immunoglobulins on HLA-specific antibody-producing cells, and an Fc effector module, which mediates cytotoxicity by mouse IgG2a.⁹¹ However, the experiment was conducted utilizing B cell hybridomas to generate an immunological mouse model, which is distinct from the conventional alloimmunization model in humans.⁹¹ Therefore, further investigations are required to validate the effect of HLA-Fc fusion on specific anti-HLA antibodies and B cells. Moreover, the impact of HLA-Fc fusion protein targets is known only for one allele of the HLA molecule and varies in different serologic types of HLA, which may not be sufficient for patients with various anti-HLA antibodies.

The lower hinge/CH2 region of the IgG heavy chain is crucial for the crosslinking of IgG with FcγRIIa and can be cleaved by the IgG-degrading enzyme from Streptococcus pyogenes (IdeS), a cysteine protease.⁹² Previous studies in mice have demonstrated that IdeS can be used in treating IgG-mediated disorders, such as HIT.⁹³ However, the therapeutic usage of IdeS is hampered due to its nonselective cleavage of IgG, which makes nearly all IgG nonfunctional and substantially raises the risk of serious infection. Recently, Lynch et al.⁹⁴ produced a recombinant protein named scIV.3-IdeS by linking the C-terminus of a single-chain variable component of an anti-FcγRIIa antibody to the N-terminus of Ide that can secure IdeS to the platelet surface due to the expression of FcγRIIa. Compared to IdeS, this recombinant protein can prevent platelets from being phagocytosed by macrophages through disassembling anti-platelet antibodies without causing the excision of nonpathogenic antibodies in the ITP mouse model. Future studies should examine the immunogenicity and function of platelets after the remedy of scIV.3-IdeS. It would be worthwhile to consider applying this therapy to other antibody-driven platelet diseases with a clear Fc-dependent etiology.

Conclusion

Immune-mediated refractoriness to platelet transfusion is a long-term and insurmountable complication in transfusion medicine, and HLA alloimmunization forms a major immunological barrier to refractoriness. Understanding the cellular and molecular mechanisms of the interaction between antibodies and platelets could contribute to the future development of novel therapeutic and preventative interventions for PTR, such as recombinant proteins targeting the antibodies themselves and blockers of antibody-driven signaling pathways. The studies on the role of CD8⁺ T cells in PTR are limited, which is a drawback to our comprehension of immune-mediated PTR. Further research on the pathophysiologic processes of alloimmunization and alternative therapies is required to give scientific proof in support of the clinical practice.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the Natural Science Foundation of China under Grant No. [82172335, 81971994, and 91846103]; Zhejiang Provincial Key Research and Development Program under Grant No. [2020C03032], and Zhejiang Provincial Natural Science Foundation of China under Grant No. [LQ20H080002].