Investigative and laboratory assays for allogeneic rejection—A clinical perspective

Qiang Shi， Afzal Nikaein（. Division of Transplantation，Department of Surgery，School of Medicine and Public Health，University of Wisconsin-Madison，Madison 5379，Wisconsin，American ；. Transplant Immunology， Medical City Dallas Hospital，Dallas 7530，Texas，American）

【編者按】實(shí)驗(yàn)室檢測(cè)對(duì)于預(yù)測(cè)和監(jiān)測(cè)器官移植術(shù)前后的耐受性具有無(wú)法估量的價(jià)值。本文討論了可用于幫助臨床醫(yī)生評(píng)估移植受者中同種異體移植物狀態(tài)的檢測(cè)方法。討論導(dǎo)致同種異體移植排斥反應(yīng)的先天和適應(yīng)性分子和細(xì)胞免疫反應(yīng)，特別是重點(diǎn)關(guān)注抗體介導(dǎo)的器官排斥反應(yīng)，以及僅在活檢樣本中發(fā)現(xiàn)的亞臨床排斥反應(yīng)。然后，從臨床的角度介紹新近開(kāi)發(fā)的器官排斥技術(shù)，總結(jié)了目前用于監(jiān)測(cè)器官排斥反應(yīng)免疫活性的方法以及未滿(mǎn)足的診斷需求和挑戰(zhàn)。最后，將提出新的方法來(lái)協(xié)助實(shí)驗(yàn)室診斷器官排斥反應(yīng)。本研究的最終目標(biāo)是提供見(jiàn)解，幫助科學(xué)家在實(shí)體器官移植后為早期診斷生成可靠的非侵入性工具，并幫助臨床醫(yī)生開(kāi)發(fā)基于預(yù)測(cè)性生物標(biāo)記物的個(gè)體化移植前和移植后治療。

T he need for organ replacement has been steadily increasing across the globe and new challenge may dramatically change the medical landscape over the coming years. According to the United Network for Organ Sharing （UNOS）， more than 33 600 organ transplants were performed in the United States in 2016. The need for organ replacement has been steadily increasing， as improved medical care， nutrition and public health continue to extend life expectancies across the globe.

In addition to reducing costs and increasing the availability of transplantable organs， it is important to control rejection rates and improve functionality of organs after transplants. New approaches， such as molecular diagnostics， proteomics and synthetic biology are being used to evaluate the status of transplants in recipients，and novel genes， small RNAs， proteins， and pathways associated with transplant rejection are being discovered at an accelerating rate1. As these discoveries are being made with ever-increasing speed， it is important to assess their clinical relevance. In addition to evaluate the sensitivity and specificity of assays to detect early allotransplant rejection signals， we must work to understand the information these assays convey to successfully translate our benchtop discoveries into bedside decisions.

In this review， we will discuss the innate and adaptive molecular and cellular immune responses that lead to allotransplant rejection. In particular， we will focus on the rejection activities that occur around the time of transplantation， as well as subclinical rejection seen only in biopsied samples. Then we will introduce newly developed technologies for organ rejection from a practical point of view. Finally， we will propose new method that will assist laboratory diagnosis in organ rejection.Our ultimate goal is to provide insights to help scientists generate reliable， noninvasive tools for early diagnosis after solid organ transplantation and assist clinicians developing individualized pre- and post-transplantation treatments based on predictive biomarkers.

1 Alloreactive Cells and Their Modes of Action

It is worth mentioning that the immunologic destruction of an allograft involves the complex interplay between the innate and adaptive immune systems2.Figure 1 lists the components that are involved in allogeneic rejection and the main characteristics of innate and adaptive immune systems. In general， tissue damage occurred during surgical organ grafting triggers innate immune system including allogeneic antigens processing by phagocyte and dendritic cells. Activation of the innate immune system initiates and amplifies the adaptive responses including naive T and B cells differentiation， while B cells require help from T cells for antibody production. Even though one component of immune system may dominate and lead to rejection， it is usually multifactorial resulting from the integration of multiple mechanisms. As it was pointed out by Thomas et al that the failure of a grafted organ is the result of a perfect and orchestrated storm of innate and adaptive immune reactions that represent by HLA antibody-mediated inflammation in the microvascular bed3.

1.1 Initial innate immune response to donor organs

The human body inherently possesses delicate molecular signals， called danger-associated molecular patterns， which promote an instant immune reaction in response to the allograft. Danger-associated molecules are immediately mobilized to act within 0 to 12 hours of recipient contact to donor organ. They are responsible for the recipient’s innate ability to sense the donor organ，the allograft. A subset of danger-associated molecules is recognized as pattern recognition receptor （PRRs）， whose members， molecular structures and function are well elucidated in the context of allograft rejection4-5. PRRs are expressed on antigen-presenting cells， including tissue resident phagocytess， dendritic cells， natural killer（NK） cells and mast cells， as well as endothelial and organ parenchymal cells. When a donor organ is grafted into the recipient， antigen-presenting cells from the donor organ may sense host-derived molecules released from damaged or stressed tissues through blood circulation，and these cells are capable of activating PRRs， leading to early rejection. Therefore， the entire body is able to sense conserved peptide sequences released from damaged graft tissue and identify invading non-self antigens from self. Although the study of PRRs originated in the context of infectious bodies， recently data suggest that these receptors are involved in organ rejection6-7.

Once allograft antigens are recognized， PRRs trigger a cascade of signaling events leading to the transport of cytoplasmic NF-κB protein complexes into the nucleus.The donor organ endothelium is particularly important for mediating downstream immune reactions， because it is the first foreign tissue encountered by the host blood cells. The outcome of this signaling transduction is a rapid increase in the transcription of genes associated with inflammation，resulting in the steady production of proinflammatory cytokines， chemokines， and adhesion molecules by activated endothelial and parenchymal cells. This early inflammatory response accelerates rejection through upregulating co-stimulatory molecule expression， which disrupts the balance between co-stimulatory and coinhibitory signals， resulting in greater tissue damaged.Therefore， an antigen-independent proinflammatory episode occurs soon after transplantation， it participates the early stage allograft rejection response8-9.Although there are many experimental tools to detect PRRs， there are few laboratory tests that enable clinicians to evaluate the status of PRR-related innate immunity. Therefore， this is one of unmet needs for clinical laboratory to evaluate innate reactivity to allografts. It is a subject that will benefit to organ allocation to a significant extent.

1.2 Identification of allo-immune cells activated during transplantation

Adaptive immunity， which follows the initial immune responses to the graft， specifically targets the allograft.Allograft-reactive （alloreactive） immune cells play a major role in the adaptive immune response， which determines to a large extent whether a transplant is accepted， rejected or becomes tolerance （Figure 1）. Basic and clinical researchers are developing methods to track these cells in live to monitor alloreactive responses， which would enable clinicians to make earlier predictions for transplant outcome. There are various techniques available to identify and quantify donor-specific T， B cells and plasma cells，we briefly outline these methods as follows.

Figure 1 Molecular and cellular components involved in allogeneic rejection

The oldest and most widely used in vitro assay to measure the adaptive immune response is the mixed lymphocyte reaction （MLR）， established in 1963. For this assay， peripheral blood mononuclear cells from the donor are co-cultured with those from the recipient， and the activation and proliferation of alloreactive T cells， as well as their cytokine production， are monitored11. This clinically relevant functional assay has been extensively optimized and standardized for reproducibility and compliance with clinical testing standards. There are a few varieties of MLR， which can determine one， two or three immune cell populations. Based on the readouts of MLR tests， they can be modified to determine different aspects of allograft rejection. For example， bromodeoxyuridine can be used to indicate proliferating cells and cytotoxicity against donor cells can be measured by sequentially diluting responder cells using a limiting dilution assay.In combination with flow cytometry or an enzyme-linked immunosorbent assay （ELISpot）， MLR enables us to precisely quantify the number of cytokine-producing cells after stimulation and determine the expression of surface molecules participating in various immune activities， such as co-stimulation， antigen presentation，cytokine production， and chemotaxis. Quantification of the cell-cycle entry marker Ki67 or intracellular carbooxyfluorescein succinimidyl ester （CFSE）， whose intensity is halved with each cell division， can also be used to measure cell proliferation. MLR largely measures the proliferation of T cells. Both naive and memory CD4+and CD8+T cells from human peripheral blood have been shown to proliferate in this test. Anywhere between 1%and 10% of the entire T cell population has been shown to be alloreactive， depending on the experimental method and sample types12-13.

However， results of MLR suggest the existence of the direct pathway of allogeneic recognition， in which T cells are activated by allogeneic antigen-presenting cells.MLR data is in contrast to in vivo scenarios， in which T cells are activated by peptides derived from donor protein molecules presented by autologous cells. This difference is critical in regard to immune recognition， as the entire major histocompatibility complex molecule behave differently from the peptides when they are presented by autologous cells， their capacities to generate different clones may vary. Currently MLR is primarily used in the pharmaceutical industry， not yet for clinical evaluation.

To provide cellular evidences for the potential clinical episodes， it is necessary to determine the exact numbers and diversity of T cell clones that are able to recognize the allogeneic major histocompatibility complex (MHC). One well-developed method for identifying alloreactive clones is next-generation sequencing， together with fluorescenceactivated cell sorting14-15. After recipient T cells react to donor cells in ex vivo culture， responsive dividing cells are sorted using carboxyfluorescein succinimidyl ester（CFSE） labeling. Genomic DNA is extracted from this dividing population， as well as from untreated recipient T cells， and high-throughput sequencing of the T cell receptor β chain CDR3 region is performed. The data are combined to generate a “fingerprint” specific to T cells for recipient reaction to the donor cells. With this technique，thousands of clones arising as a result of the allogeneic reaction can be identified in the peripheral circulation after transplantation. Several groups are investigating their clinically-relevant response by the use of this method.

Usually， the segments of MHC molecules of interest can be synthesized to form peptide MHC multimers. When labeled with a fluorophore， these oligomers can bind target MHC specifically， enabling the polyclonal alloreactive cells to be traced to a single donor antigen. Due to their high specificity and reproducible results， peptide MHC multimers have become one of the most widely used tools to measure antigen-specific T cell responses in mice.Before this technique may be used in humans in the future， there are still several challenges to address. The design of peptide MHC multimers requires the selection of a peptide that alloreactive cells can recognize， and individuals may express subtypes of the commonly studied HLA alleles， limiting the use of this method for the time being. Another drawback to the use of peptide multimers for functional studies is the kinetics of peptide binding to the receptors on the cells， as it may alter T cell proliferation and exhibit effector function. For example， T cells bind naturally processed antigens for an average of 7 seconds in vivo but can bind synthetic peptides for as long as 967 seconds. The ways in which this prolonged binding may affect downstream signal transduction has not been thoroughly assessed. Reversible forms of peptide MHC have been used， but the analysis of alloreactive responses remain to be revealed.

Studies on in vivo stimulation of allogeneic cells is another approach for identifying alloreactive T cells，because cell culture conditions are not fully optimized for some types of lymphocytes. The trans-vivo delayed hypersensitivity model is a current assay that allows us to study of allogeneic rejections using patient samples16-17.The authors isolated peripheral mononuclear cells from the recipient and prepared lysed donor cells. Then，they injected a mixture of these cells into the footpad of a severe combined immunodeficiency （SCID） mouse.After 24 hours， the injection of alloreactive mononuclear cells resulted in greater swelling and redness than the control injection of mononuclear cells alone. This reaction indicated that the patient developed an immune memory to the donor antigen presented through the indirect pathway，because the lysate lacks intact donor antigen-presenting cells. The advantage of this assay is the polyclonal nature of alloreactive process， which is closer to the reality of organ transplant fate in the recipient. Although the in vivo assay has advantages over in vitro and ex vivo models，stimulation by these methods may not reflect the actual fate of donor antigens during organ transplantation， when stimuli are derived and processed from an engrafted tissue over months to years.

Peptide MHC tetramer-based system has also been used to measure the expansion and differentiation of antigen-specific B cells after sensitized by transplantation.After initial stimulation by alloreactive antigens， B cells undergo a process of class switching， during which they are activated and differentiate into antibody-secreting cell ex vivo. Class switching can be measured through IgG ELISpot assays or by the assessment of secreted IgG in the supernatant. Recently， some groups have used microspheres coated with HLA molecules to bind HLA-specific B cells bind， forming HLA bead-B-cell rosettes that can be identified by flow cytometry. With this approach， the identity and frequency of B cells specific for HLA alleles， as well as their phenotypes， can be rapidly determined in a time-frame comparable to donor-specific antibody assays18.

The primary culture of B cells， the antibody producers， is more difficult to maintain than the culture of T cells， and in vitro B cells assays have not long been established. One solution to overcome the challenges of primary B cell culture is to use feeder cells that promote B cell proliferation and differentiation via cell-cell contact.Using this method， memory B cells can differentiate into terminal stage （long-lived plasma cells）， since they are the major population that produces allogeneic antibodies for antibody-mediated organ rejection16.

Plasma cells are another cell type that remains difficult to track， because they do not express suitable markers—they express little to no CD19 or B cell receptors.Though CD138 is a marker， it is expressed by other cell types， thus lacking specificity. The lack of marker complicates the identification of plasma cells by flow cytometry. An ELISppt assay that immobilizes anti-IgG and biotinylated MHC monomers was recently used to detect the frequency of plasma cells secreting MHC-specific antibodies. Using this method， the authors found that plasma cells increased 104-fold in the spleen and 103-fold in bone marrow before and after transplantation. These tissue resident， long-lived plasma cells are considered responsible for serological memory and are the main sources of donor-specific antibody production. Once plasma cells are identified， there is considerable interest in developing desensitization protocols that can eliminate these cells in patients with high reactive antibodies at high risk for rejection16-18.

2 Allogeneic Antibodies：Characteristics and Clinical Relevance

Over the past decade， the pharmaceutical industry has produced effective immunosuppressing drugs that can prevent or treat T cell mediated rejection19-20. Therefore，acute rejection that occurs via cellular immunity within days or months of transplantation has been well controlled in the majority of recipients. Most allografts are lost due to antibody mediated rejection （AMR） that often begins several months after grafting. Most laboratories rely on solid phase assays， such as immunoassays on the Luminex bead platform， for antibody detection. In clinical practice， panel reactive antibody （PRA） tests are routine pre- and post-transplantation. Calculated PRA （cPRA）， a corrected ratio of PRA frequency based on local population， is used to indicate the likelihood of experiencing an acute rejection to a local donor’s tissue pool. In addition， pre-transplantation PRA is becoming a part of virtual crossmatch.

2.1 AMR and its characteristics

AMR is a dynamic process that occurs through various mechanisms. It is generally agreed that acute rejection depends on components of the complement system， whereas chronic rejection does not21-22.Figure 2 presents mechanisms that underlie AMR， especially those de novo donor specific antibodies that participate in the induction of antibody mediated organ failure.In the initial alloreactive responses， HLA and non-HLA antigens activate T cells and present processed antigens to B cells that eventually produce antibodyproducing clones. These antibodies vary in specificity，affinity for complement proteins， isotype/subclass，strength （MFI）， density and glycosylation11，23.When de novo donor-specific or non-HLA antibodies are able to fix complement proteins such as IgG1 and IgG3， they tend to activate complement complexes and deposit their C3d or C4d fragments around microvessels.Complement-fixing antibodies also have positive reactions in C1q binding tests. However， some types of antibodies，such as IgG2 and IgG4， lack complement fixing abilities and do not cause complement deposition on microvessels24-25. In acute rejection， donor specific antibodies cause graft damage through complement activation via Fc receptors. After prolonged exposure and de novo production of donor specific antibodies，pathogenic antibodies act on the surface of the endothelium through coupling and aggregating allogeneic HLA molecules and activate signaling pathways26-27.

Figure 2 Mechanisms underlying antibody mediated rejections

Cross-matching is a well-established laboratory method to detect complement-fixing antibodies based on their ability to lyse lymphocytes placed on a microtiter plate. However， there is no equivalent method to detect the early signs of chronic rejection that occur in the microvascular bed. Figure 3 illustrates the pathophysiological consequences upon grafting， and manifestations that can be monitored in the laboratory to detect allogeneic antibody mediated graft failure. Donorspecific antibodies， C4d expression and biopsy tissue abnormalities， including microvascular inflammation and its chronic sequelae， transplant glomerulopathy， and basement membrane multilayering. Delayed graft failure roots its origin from microvascular inflammation， which significantly affects overall patient outcome28-30. The current approach for pathological evaluation is biopsy，but it is expensive and impractical. Moreover， bloods test for rejection， such as PRA， may provide ambiguous results， as discussed earlier in this manuscript. It is imperative to develop an assay capable of accurately and specifically diagnosing the occurrence of rejection in transplanted patients.

Figure 3 The pathophysiology of graft rejection and detectable alterations for clinical diagnosis and prognosis.

2.2 Discrepancies between antibody repertoire and graft outcome

Although PRA is the most widely used predictive test for antibody mediated rejection， the degree of sensitization represented by PRA is highly variable and inconsistent. The antibody status of a recipient does not precisely correspond to recipient transplantation milieu31-32， and it is not reliable for selecting compatible donors or comparing relative degree of sensitization33.What has been discovered about alloreactive antibodies so far is that not all antibodies are created equally34-35.Their functional or clinical implications vary greatly.A widely used concept is pathogenic antibody， which distinguishes the repertoire of allogeneic antibodies. At the molecular level， a pathogenic antibody binds to epitopes that may activate cellular signaling pathways and lead to a biological response. Allogeneic antibodies are only detrimental if they possess the ability to mediate allograft injury and contribute to pathological alterations through reactions， such as complement activation， leukocyte adherence， endothelial activation and microvascular proliferation. Some antibodies may not be harmful at all if they bind to a non-activating epitope. There are increasing papers about the studies on epitope biology， and how interactions between epitope antibodies impact on solid organ transplantation19，36-38. The recognition of a hierarchy of allogenic antibody responsiveness to HLA epitopes may reveal the solution for clinical AMR.

To bridge the difference between antibody nature and clinical outcome， we will briefly recall how B cells produces antibody. Kosmoliaptsis and colleagues have presented an excellent paper detailing the relationship between the high-resolution， three-dimensional architecture of HLA molecules and their alloantibody binding characteristics39. Wherever recognized by naive B cells， the particular three-dimensional structure may elicit a clonal B cell line development that produces specific antibody against this region. Collectively， these regional areas are called “epitopes”. Due to the large size of HLA molecules， a recipient’s immune cells only recognize the epitopes rather than react to the entire HLA molecule40. There is virtually no any antibody that can react to any entire HLA molecule but against specific epitopes. In other words， one mismatched HLA molecule can potentially give rise to numerous clones against their corresponding sites. As a matter of fact， it is the repertoire of these antibodies that determines the recipient’s ability to tolerate the allograft. To date， several algorithms have been developed to discover the peptide-epitopes to predict the AMR. It will soon be possible to determine all epitopes on every HLA molecule， which will be a significant advancement for the exact characteristics of B cell clone41.

2.3 Shortcomings of solid phase bead assays

During 1990s， solid phase detection was revolutionized by the introduction of fluorescently labeled bead technology and became the most widely used method of HLA antibody detection， as Luminex-based technology.In this method， one or more different types of synthetic human HLA antigens are immobilized on beads. If the serum from the patient contains an HLA antibody， it will bind to corresponding bead so the machine can detect it.The bead not only indicates the type of antibody present in the serum， but also tells us the “l(fā)evels of antibody” based on changes in mean fluorescence intensity （MFI） based on the saturation of antibodies on the bead surface. More antibodies circulating in blood leads to higher saturation of antigen-antibody binding on the bead surface42.

This method is very sensitive and specific， but after many years in the clinical setting， it has become clear that a substantial number of patients with chronic AMR have no detectable circulating HLA antibody at the time of diagnosis. The details of this finding were described in a 2013 report under the guidance of the Transplantation Society. Several recent review papers describe the theoretical and realistic reasons why there are considerable differences between laboratory test and clinical outcomes. As Filipone explained， solid phase bead assays detect only the alloreactive antibodies that can combine stereotypically with the immobilized epitopes designed by the manufacturer41. If the binding region of analloreactive antibody does not fit the epitope of the target HLA that has been coated on the bead， it will generate a negative result42-43. Historically， information about various antibody alleles does not exist before they are found to have caused an adverse immune response. Manufacturers who produce single bead for allogeneic antibodies rely on synthetic peptides that represent epitopes rather than entire structure of an HLA molecule. Unfortunately， there is little information to estimate how much this technology has biased clinical outcomes due to a clinical based systematic study. In addition， the binding of antibodies to antigens on beads has no functional significance， so the results of these assays do not enable us conclude whether the antibodies detected have pathological consequences.In other words， this test can only suggest a status of patient sensitization but cannot be used to predict actual outcomes.The lack of factional aspect of the test also explains some inherent issues associated with this method， whenever interpret sensitivity， fluorescence cut-off and denatured HLA. These questions are associated with the design of this test that remain to be solved in order to directly relate to pathogenicity of alloreactive antibodies.

For the above reasons， it is important to develop test that enable us to distinguish pathogenic antibodies from non-pathogenic antibodies. As we mentioned earlier，pathogenic antibody exerts its role not only by binding the corresponding epitopes on the antigen but also elicits subsequent biological reactions.They can cause rejection via two possible routes via Fc-mediated or Fab-mediated actions， as shown in Figure 2. Approximately 20% of stable patients with no evidence of rejection on biopsy were positive for donor-specific antibodies. Up to half of patients with preformed donor-specific antibodies did not have rejection， nor subclinical AMR at the time of biopsy. However， rejection can occur in patients with a low donor-specific antibody titer and donor-specific antibody levels do not correlate neatly with the incidence of rejection. These issues can be possibly resolved with the use of functional antibody test. The development of such tests will provide supplemental data for evaluation of organ rejection to the utility of Luminex-related data.

2.4 New laboratory assays for the detection of AMR

To address the need for functional test， we are developing an innovative cell-based assay. We have designed a reconstructed cell tray made of human cells that enables us to mirror the intricate interactions between recipient humoral factors and the graft vascular bed in vivo. Laboratory investigations have shown that incubating panel reactive antibodies with endothelial cells causes them to become activated and dysfunctional38，44-45.Further investigations indicate that HLA antibodies and non-HLA antibodies can act as agonists to introduce intracellular signaling. The inflammation and proliferation of transplanted tissue is the most important determinant of allogeneic organ survival11，19. We are making efforts to translate the laboratory findings to appropriate techniques that enable us to quantitatively measure. Our assay development bases on the fact that activated endothelial cells release proinflammatory cytokines and express high levels of adhesion molecules on the surface. These are the molecular basis for the infiltration of macrophages and lymphocytes， pathologically defined as vasculitis or microvascular inflammation， which represents one of the earliest events of allo-rejection.

Functional testing for donor specific antibodies impacts the clinical practice of organ transplantation.For the time being， biopsy is the only method for diagnosis， and no other laboratory resource is available to detect AMR. The current diagnosis of AMR depends on Banff classification based on cellular and/or structural alterations； however， these classifications are considered“presumptive” not decisive. In addition， there is no definitive approach to diagnose subclinical AMR33，46.Our proposed assay can potentially mirrors the status of the interaction between the recipient humoral immune response and the transplanted vascular bed， and interrogate the health of transplanted organs with enhanced accuracy and precision. This method not only detects the occurrence of endothelial activation， but also quantifies the degree of the interaction between recipient sera and donor cells， which may provide the earliest signs of organ rejection21-22. Therefore， the test results from this assay can provide a window for clinicians to surveillance transplanted organ health and detect the onset of transplant rejection， without the need for biopsied samples.

3 Acknowledgments

This work was supported by Tianjin Science and Technology Commission （17ZXSCSY00100）.