# ANTIBODY REPERTOIRE AND GRAFT OUTCOME FOLLOWING SOLID ORGAN TRANSPLANTATION

EDITED BY: Narinder K. Mehra, Ajay Kumar Baranwal and Brian D. Tait PUBLISHED IN: Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88945-241-5 DOI 10.3389/978-2-88945-241-5

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# **ANTIBODY REPERTOIRE AND GRAFT OUTCOME FOLLOWING SOLID ORGAN TRANSPLANTATION**

#### Topic Editors:

**Narinder K. Mehra,** All India Institute of Medical Sciences, India **Ajay Kumar Baranwal,** All India Institute of Medical Sciences, India **Brian D. Tait,** Australian Red Cross Blood Service Australia, Australia

Cover image designed by Mr Ajith

The first real major breakthrough that laid the basis of HLA antibody detection in the field of solid organ transplantation, came with the introduction of the complement dependent cytotoxicity (CDC) test in 1964 by Terasaki and McClelland. Since then, methods for antibody detection have evolved remarkably from conventional cellbased assays to the current advanced solid phase systems on the Luminex platform, with increasing degree of sensitivity and specificity. The latter have been indispensable for more accurate identification of donor specific HLA antibodies in broadly reactive allo antisera, and to guide donor selection and kidney paired exchange programs through virtual crossmatching, in addition to serving as excellent tools for initiating pre-transplant desensitization and post- transplant antibody monitoring. Consensus is evolving on the optimal routine employment of these methods in donor selection

strategies along with an understanding of the clinical relevance of antibodies detected by each of them.

The immunoassays based on the Luminex platform and flow cytometric beads are however unable to discriminate complement fixing from non-complement fixing HLA antibodies. This is important because the former are considered clinically more pertinent in the peri-transplant period. The C1q assay which is a modification of the solid phase assay based on Luminex single antigen beads, which can be used effectively to monitor high dose IVIG desensitization is essentially a surrogate complement fixing assay, retaining the exquisite sensitivity and specificity of the Luminex platform. Currently, information obtained from these assays is preliminary and much needs to be done to standardize technologies and set a consensus 'MFI cut off' for antibody positivity.

Besides the overriding influence of anti-HLA antibodies on overall solid organ graft survival, immune response to non-HLA antigens has become a topic of substantial interest in recent years. An ever expanding list of non-HLA antigens has been implicated in graft rejection for various organs, of which the most noted are the Major Histocompatibility Complex class I chain-related molecule A (MICA), Vimentin, Myosin, Angiotensin II type 1 receptor (AT1R), Tubulin and Collagen. MICA is one of the most polymorphic and extensively studied non-HLA antigenic targets especially in renal transplantation. Although there are clear indications of MICA antibodies being associated with adverse graft outcome, to date a definitive consensus on this relationship has not been agreed. Because MICA molecules are not expressed constitutively on immunocompetent cells such as T and B lymphocytes, it is of utmost importance to address the impact of MICA donor specific antibodies (DSA) as compared to those that are non- donor specific (NDSA) on graft outcome.

The soluble isoform of MICA molecule (sMICA) that is derived from the proteolytic shedding of membrane bound molecules has the potential to engage the NK-cell activating receptor NKG2D and down-regulate its expression. Consequent to the interaction of NKG2D by sMICA, the receptor ligand complex is endocytosed and degraded and thus suppresses NKG2D mediated lysis of the target by NK cells. Thus interaction between NKG2D and sMICA leads to expansion of immunosuppressive/anergic T cells thereby resulting in suppression of NKG2D mediated host innate immunity. These concept support the possible involvement of an immunosuppressive role for sMICA during allotransplantation as shown recently for heart transplantation.

This research topic focusses on the clinical utility of investigating the complete antibody repertoire in solid organ transplantation.

**Citation:** Mehra, N. K., Baranwal, A. K., Tait, B. D., eds. (2017). Antibody Repertoire and Graft Outcome Following Solid Organ Transplantation. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-241-5

# Table of Contents

#### **I. Editorial**

#### *128 Reflections on HLA Epitope-Based Matching for Transplantation*

Rene J. Duquesnoy

**IV. Cellular Aspects of Antibody Responses**

*136 Donor-Specific Regulatory T Cells Acquired from Tolerant Mice Bearing Cardiac Allograft Promote Mixed Chimerism and Prolong Intestinal Allograft Survival*

Xiao-Fei Shen, Jin-Peng Jiang, Jian-Jun Yang, Wei-Zhong Wang, Wen-Xian Guan and Jun-Feng Du

*146 B Cell Immunity in Solid Organ Transplantation*

Gonca E. Karahan, Frans H. J. Claas and Sebastiaan Heidt

*157 The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation*

Yongkang Wu, Nicole M. van Besouw, Yunying Shi, Martin J. Hoogduijn, Lanlan Wang and Carla C. Baan

*167 The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection*

Raja Rajalingam

# Editorial: Antibody Repertoire and Graft Outcome following Solid Organ Transplantation

*Narinder K. Mehra 1 \*, Ajay Kumar Baranwal 1 and Brian D. Tait 2*

*1All India Institute of Medical Sciences, New Delhi, India, 2Australian Red Cross Blood Service Australia, Melbourne, VIC, Australia*

Keywords: antibody, graft, organ transplantation, anti-HLA antibodies, sMICA

**The Editorial on the Research Topic**

#### **Antibody Repertoire and Graft Outcome following Solid Organ Transplantation**

In recent years, there has been vast improvement in the survival of solid organ grafts. Improved immunosuppression has resulted in efficient control of cell-mediated immune responses and has also mitigated the effects of HLA mismatching between the recipient and the grafted organ.  Accurate and sensitive HLA antibody identification has been made possible by the introduction of solid phase assays such as the Luminex bead based platform for antibody testing.

Despite this advancement in detecting and understanding the role of HLA antibodies in rejection, many questions still require answers in order to maximize the outcome in solid organ transplantation. Many of these aspects are dealt with efficiently in this excellent series of papers addressing the role of antibodies in solid organ rejection.

This collection of reviews, some including new findings, by world leaders in their particular subspecialty, represents a compendium of the most recent and exciting developments of special interest to those who are involved in understanding the role of antibodies in organ rejection.

The technical aspects of antibody detection have been addressed in detail by Tait (Melbourne). While solid phase and, in particular, the Luminex bead assays have revolutionized the way antibody screening is now conducted, they have produced their own particular challenge. The failure to discriminate complement from non-complement fixing antibodies by solid phase assays poses a problem with respect to the significance of a positive result, which has been partially addressed by modification of the Luminex assay to measure complement fixation.

Data from clinical studies suggest that non-complement fixing antibodies may compromise graft outcome, albeit not to the same extent as complement-fixing HLA antibodies. The presence of antibodies directed at denatured HLA and detected by the Luminex platform, although not clinically important can also complicate the analysis of antibody specificity determination. Issues such as the importance of the mean fluorescence index on graft outcome and the value of the virtual cross match are also discussed in this review.

The functional aspects and clinical impact of alloantibodies and autoantibodies are discussed in several excellent reviews.

Despite the best efforts of avoiding pretransplant HLA antibodies, there is no way of predicting which patients will develop *de novo* antibodies. Mangiola and co-workers (Pittsburgh) discuss the role of both pretransplant and *de novo* HLA antibodies in pediatric and adult heart transplant recipients. They stress that the treatment for early and late rejection are different, which raises the

#### *Edited and Reviewed by:*

*Antoine Toubert, Paris Diderot University, France*

> *\*Correspondence: Narinder K. Mehra<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

> *Received: 04 May 2017 Accepted: 17 May 2017 Published: 09 June 2017*

#### *Citation:*

*Mehra NK, Baranwal AK and Tait BD (2017) Editorial: Antibody Repertoire and Graft Outcome following Solid Organ Transplantation. Front. Immunol. 8:648. doi: 10.3389/fimmu.2017.00648*

important question of the optimal protocol for posttransplant screening in order to maximize timely detection of antibodies prior to organ damage.

In addition to HLA antibodies, the role of non-HLA antibodies is discussed by several author groups. Matsuda and Sarwal (San Francisco) emphasize the point that antibody-mediated chronic rejection remains the biggest unresolved issue. Chronic AMR has been shown in cases involving HLA identical recipients and donors suggesting a role for non-HLA antibodies. This review is a "tour de force" covering mechanisms underlying antigen recognition, the role of both HLA and non-HLA antibodies in rejection and immunosuppressive approaches.

Nayak et al. (Phoenix) studied both auto- and alloantibodies in lung and pancreatic islet cell transplantation, which are more susceptible to rejection by a combination of antibodies than other forms of transplants. Islet cell transplant patients are often exposed to multiple islet cell infusions and as a result are exposed to many HLA class 1 and 2 mismatches. In addition to HLA immunity, there is evidence that autoantibodies to GAD65 and autoreactive T cells participate in the rejection process. It appears that once tolerance is broken to the self-antigens, then immunosuppression becomes ineffective. In lung transplantation, HLA antibodies are associated with obstructive airway disease while the appearance of MICA antibodies after HLA donor-specific immunization is associated with bronchiolitis obliterans syndrome.

Zhang and Reinsmoen (Los Angeles) have submitted a comprehensive review on the role of non-HLA antibodies in kidney and heart transplant rejection. Antibody specificities discussed include those to myosin, vimentin, Kα1 tubulin, collagen, and angiotensin II type 1 receptor. There appears to be synergy between HLA and non-HLA antibodies. In some cases, development of HLA antibodies precedes the development of antibodies to non-HLA antigens. It is suggested that the damage inflicted on the graft by HLA antibodies exposes cryptic antigens on non-HLA molecules that result in antibody formation by the recipient. There is also evidence that the presence of non-HLA antibodies may predispose the recipient to the formation of HLA antibodies. Given this close relationship between HLA and non-HLA antibodies, the authors stress the point that it is imperative that both types of antibodies be measured in transplant recipients.

Baranwal and Mehra (New Delhi) discuss the importance of MICA antibodies on graft outcome in a thorough review of the literature and demonstrate that MICA antibodies are detrimental to the outcome of solid organ transplantation, but soluble MICA appears to have an inverse relationship to rejection. The mechanism behind this observation appears to be the interaction of the soluble MICA with the MICA ligand NKG2D, thus blocking the activation of NK cells. Amino acid position 129 appears to be pivotal in the induction of immunity in renal transplant patients. Recipients who are homozygous for methionine at this position have a higher incidence of rejection than those with valine homozygosity. The mechanism underlying this association is not understood.

Valenzuela et al. (Los Angeles) discuss the impact of IgG subclass on solid organ rejection. The histology and clinical profiles surrounding both HLA and non-HLA donor-specific antibodies is very heterogeneous with limited understanding of the various roles that the IgG antibody subclasses play. The authors describe in detail the various functions that IgG subclasses subserve, the data for which is largely derived from infectious disease studies and human cancers, but make the point that in clinical transplantation there is little known. Analyzing all known data suggests that IgG3 is predominantly associated with complement-mediated rejection while IgG4 is associated with memory, and subclinical and chronic rejection.

One of the complicating issues is that most patients have mixtures of two or more isotypes, which makes analysis of single isotype function difficult. Newer assays are needed that could measure single antibody isotypes, permitting correlations to be established between antibody isotypes and the various features of complex rejection histology.

An intriguing piece of original research by Geneugelijk et al. (Basel) focuses on nature's allograft, the fetus. They demonstrate that a previous miscarriage prior to a successful second pregnancy produces a lower rate of immunization to paternal HLA than a previous full-term pregnancy. Examination of the number or epitope differences between the mother and fetus revealed the intriguing observation that patients with a previous miscarriage actually had a lower rate of HLA incompatibility. This observation is not readily explained, although the authors have put forward several hypotheses that require further exploration. Suffice to say the mechanism underlying this observation may have some relevance to immunization mechanisms in the clinical allograft situation.

Rajalingam (San Francisco) discusses the role of NK cells in organ rejection. At first sight, this review does not seem central to the theme of the series. However, the link is made by virtue of the fact that the presence of HLA antibody bound to graft endothelium can activate NK cells *via* the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway. Infiltration of grafts with NK cells have been shown in renal, cardiac, liver, and lung transplants indicating a key role for these cells in the rejection process, and NK cell transcripts have been demonstrated in kidney biopsies undergoing AMR.

Morath et al. (Heidelberg) provide an excellent review of ABO incompatible (ABOi) renal transplants with data from the Collaborative Transplant Study (CTS) established by Gerhard Opelz in Heidelberg in 1982. This has been an invaluable resource over four decades comprising 400 participating transplant centers from 42 countries, providing data on kidney, heart, lung, liver, and pancreas transplants.

The concept of a major ABOi renal transplant was an absolute contraindication. However, over the last 25 years, the need to expand living donor options for some patients led investigators into the possibility that with ABO antibody reduction pretransplant, it was possible to transplant across the major ABO barrier. If the ABO titer can be reduced to below 1:32 by plasmapheresis, membrane filtration or immunoadsorption, as measured by the tube method, then although there is a rebound effect posttransplant, it is nondamaging to the graft, a process termed accommodation. Antibody ablation is accompanied by other procedures designed to blunt the rebound response. These include the use of IVIG to modulate the recipient's immune system and anti-CD20 (rituximab) therapy to reduce the B cell pool. A new approach has been the use of eculizumab, a hybrid monoclonal that is a terminal complement blocker, in this case designed to block the damaging effect of the ABO antibodies bound to the graft endothelium. Results to date however are inconclusive with respect to the efficacy of this approach. A CTS study of 1420 ABOi renal transplants from multiple transplant centers has revealed that while graft survival of ABOi are comparable to ABO compatible grafts, early rejection and the complications of early infection are increased, with one additional patient per 100 dying from this complication in the ABOi.

Interestingly within the ABOi group, the use of anti-CD20 appears to have a significant beneficial effect. Although the use of ABOi transplants has greatly increased the living donor choices for many patients, the authors feel that caution has to be exercised in the use of ABOi due to the complications of infection.

The functional role of B cells has been elegantly addressed by Karahan et al. (Leiden) who stress the point that B cells play other roles in addition to their antibody-producing function. They discuss in detail the mechanisms underlying the role B cells play as cytokine producers and as antigen-producing cells as well as their ability to organize tertiary lymphoid tissue. B cells also invoke T cell immunity and of course regulatory B cells are central to control the immune response. Their pivotal message is that ablation of B cells can be detrimental as well as beneficial. Understanding how the different populations of B cells interact could lead to more rational and targeted immunosuppression.

Wu et al. (Sichuan) discuss the role of IL-21, which is produced by CD4+ T cells and their interaction with both plasma and memory B cells. As with the Karahan et al. paper, the possibility arises that these findings could be used to establish novel immunosuppressive approaches.

Two reviews discuss both historical aspects of HLA matching in solid organ transplantation and the evolution in our understanding of the epitopes recognized by HLA-specific antibodies. In the first such paper, Zachary and Lefell (Baltimore) provide an insight into the different features of HLA matching, focusing on the decreasing effect of matching influence over recent decades. A more sophisticated approach to matching is now employed, which does not treat all mismatches as equal, but rather considers epitope mismatches in the context of the patients' HLA immunological profile including desensitization procedures. When HLA mismatches present in a first donor and are repeated in a patient having a second transplant (repeat mismatches), it appears that they are only associated with increased risk of graft loss in patients who are HLA sensitized or those recipients who underwent nephrectomy of the first failed graft. It appears that reexposure to HLA class 2 is more damaging than class 1.

Rene Duquesnoy (Pittsburgh) summarizes the historical aspects surrounding the development of the epitope and eplet concept of HLA antibody recognition, for which he has been the preeminent pioneer. The original concept of treating each HLA serologically defined antigen as a single entity was overturned by the demonstration using sequence data, that each HLA molecule consists of multiple epitopes, some unique, and some shared with other antigens. The approach that arose from this realization was the use of epitope data in the selection of organ donors, particularly for sensitized patients but also recognizing permissible HLA mismatches in non-sensitized patients. In this excellent review, we are taken on a journey from the initial serological demonstration of antibodies, which broadly recognized HLA antigens, to the study of these antibodies at the molecular level, and the demonstration that there are polymorphic triplets of amino acids which are the signature of the epitope and are referred to as eplets. As this concept gains wider acceptance among transplant units, HLA epitope matching will become the method of choice, replacing the historical method of HLA antigen matching currently in use in many centers.

The final contribution, despite being tangential to the topic under discussion, provides some insight into allograft tolerance in a mouse model. Shen and coworkers (Nanjing, China) utilized a mouse cardiac transplant model to generate T regulatory cells and then transfer them to an intestinal transplant model where they observed prolonged survival. The use of T regulatory cells with costimulation blockade appeared to be a successful tolerance model.

In summary, although great progress has been made in the detection of HLA and non-HLA antibodies and their relevance to the outcome of solid organ transplants, there are still challenges ahead. The definition of a hierarchy of alloresponsiveness to HLA epitopes, which is dependent on the HLA molecules expressed by the recipient, may be revealed by the full sequencing of both recipients and donors made possible by next-generation sequencing. Such an approach used in a large collaborative study could make possible immunogenicity grading of epitopes interpreted in the context of individual recipients' HLA genotypes.

The expansion of antibody screening bead technology to include both HLA and non-HLA epitopes will allow further study of the detrimental or otherwise effect of various classes of antibodies. Although many of the non-HLA antibodies are considered auto in nature, an extensive study of the sequences of these genes in populations may reveal epitopes, which include polymorphic residues thus creating the possibility of using this information to select the most appropriate donor for each recipient.

Finally, many transplant recipients display mixtures of IgG isotypes, both complement and non-complement fixing. The mechanism by which mixtures of these antibodies influence rejection *via* both complement binding mechanisms and by ADCC and how these relate to both acute and chronic antibody rejection requires further study to fully understand the subtleties of these antibody interactions.

We recommend that all those interested in the role of antibodies in their many forms on the outcome and survival of a range of allograft types take advantage of this free online series of papers. They are contributed by leaders in the field of clinical transplantation and represent an up to date summary of the current state of play. Both HLA and non-HLA antibodies are discussed in the context of AMR and the mechanisms, whereby these antibodies compromise the outcome of the graft that are discussed.

The editors are pleased to be able to present this collection of papers to you as a series and feel that it represents an important contribution to the current literature on this topic.

#### AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

#### FUNDING

The authors (NM and AB) thank the Indian Council of Medical Research (ICMR) for their financial support.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Mehra, Baranwal and Tait. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The*  *use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

#### *Brian D. Tait\**

*Clinical Services and Research, Australian Red Cross Blood Service, West Melbourne, VIC, Australia*

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzymelinked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the "gold standard" for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the "virtual crossmatch" whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques.

#### Keywords: HLA antibody, CDC, ELISA, Luminex, beads, transplantation

#### INTRODUCTION

Rejection of solid organ allotransplants can be cellular or antibody mediated. In the majority of cases the rejection reaction is directed at human leukocyte antigens (HLAs) expressed on the cells of the transplanted organ. While there is no routine test which can be applied to determine the cellular immune status of potential transplant recipients, the detection of HLA antibodies, particularly those directed at the HLAs of the donor has been at the forefront of donor–recipient histocompatibility testing since transplantation became a clinical reality.

#### *Edited by:*

*Gilles Blancho, University of Nantes, France*

#### *Reviewed by:*

*Stanislaw Stepkowski, University of Toledo, USA Anne Cesbron, Blood Bank, French Southern Territories*

> *\*Correspondence: Brian D. Tait*

<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

*Received: 22 August 2016 Accepted: 23 November 2016 Published: 09 December 2016*

#### *Citation:*

*Tait BD (2016) Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay. Front. Immunol. 7:570. doi: 10.3389/fimmu.2016.00570*

The determination of antibody status is one of the most important investigations that is undertaken in potential organ transplant recipients. While levels of HLA incompatibility can be tolerated due to the quality of immunosuppressive drugs that are now available, the presence of antibodies in the recipient specific for HLA incompatibilities present in the donor can be devastating to the graft.

The first organ transplanted on a routine clinical basis was the kidney and a great deal of lessons we have learned about the impact of HLA antibodies on transplanted organs was learnt during the formative years of clinical renal transplantation.

The pretransplant crossmatch which involves testing the recipient's serum for cytotoxicity against the donor cells (lymphocytes) was introduced into the testing algorithm in the 1960s in the early days of renal transplantation (1, 2). The test which relies on the detection of complement-dependent cytotoxicity (CDC) is performed in small microtiter trays. The patient's serum and donor cells are mixed together, rabbit serum as a source of complement is added and lysis due to antibodies in the recipient specific for the donor cells is detected. The crossmatch test is still an essential component of immediate pretransplant testing for all organ transplants and is known as the microlymphocytotoxicity test. A modified form of this test was also used to screen patients' sera for HLA antibodies and to determine specificity. This method with modifications was the basis of HLA antibody screening for nearly three decades but has been replaced in recent years with more sensitive and reproducible assays of antibody activity. The evolution of HLA antibody testing and the associated laboratory and clinical issues that have arisen with the use of this new technology forms the basis of this review. Although renal transplantation is the basis for many of the lessons we have learned using the new methods of antibody detection, they apply equally to other forms of solid organ transplantation.

# HLA ANTIBODY DETECTION ASSAYS

#### Complement-Dependent Cytotoxicity

The clinical importance of the pretransplant crossmatch and the technology for performing the test was described by Terasaki and colleagues (1, 2) and became known as the microlymphocytotoxicity assay or CDC. Essentially, the test consists of incubating patient serum with potential donor lymphocytes to establish if the recipient has donor-specific HLA antibodies (HLA-DSA). Rabbit serum as a source of complement is added and if HLA-DSA are present lysis of the cells occurs. This lysis can be detected by the original method of dye exclusion or by later developments which included fluorescence. It was quickly appreciated that renal transplant patients with DSA had early hyperacute rejection (3, 4). This test was quickly established as an essential and non-negotiable pretransplant test, a negative result enabling renal transplantation to proceed.

Modifications to the test were made to make the test more sensitive such as prolonged incubation and the use of a second antibody such as an anti-IgG reagent (5) but there remain several technical problems with the test. The assay relies heavily on the viability of the donor cells and in the case of deceased donors optimal viability is not always achievable. In addition to IgG antibodies, the test detects IgM as well as auto antibodies. The latter can be overcome to some extent with the use of 1,4-dithiothreitol (DTT) (6, 7), although this can result in the loss of some IgG antibody (8).

In its original form, the assay was performed using unseparated lymphocytes from peripheral blood, lymph node, or spleen obtained by a gradient separation technique (9). This resulted in the detection of both HLA class I antibodies which react with B and T lymphocytes and also with HLA class II antibodies which react with the class II expressing B cells.

The introduction of cell separation techniques such as ficoll gradient separation (9) with subsequent rosetting T lymphocytes with sheep red blood cells (10) and then later the use of magnetic beads specific for each cell population (11) enabled the distinction to be made between HLA class I and class II antibodies. Other approaches which had varying success were also used.

The main issue with the CDC assay is its sensitivity. The development of more sensitive solid phase assays for antibody detection has basically replaced the CDC approach, but because it is the only functional assay it is still used in many centers as a final test of pretransplant compatibility in the form of the CDC crossmatch. However, even this test is slowly being replaced by the "virtual" crossmatch (see later section).

The CDC assay was modified as an antibody screening technique by using a panel of HLA typed cells and testing each patient's serum against this panel. The technique is essentially identical to the crossmatch procedure but by using a panel of cells it is possible to determine the HLA specificity of antibodies present. By testing against both T (which express HLA class I) and B lymphocytes (which express both HLA class I and II) it is possible to characterize both class I and class II antibodies when they occur together. An added step of absorbing sera with platelets, which express HLA class I but not class II, prior to testing enables the determination of class II antibody specificity without the added complicating factor of co-occurring class I antibodies (12).

By using a panel of accurate HLA phenotyped cells, it is possible to express the result as a panel reactive antibody (PRA) percentage (i.e., the percentage of cells in the panel giving a positive result) in addition to determining HLA antibody specificities. The PRA is a useful indicator of the probability of a patient giving a negative crossmatch with an unrelated donor.

# Flow Cytometry

The flow cytometry crossmatch (FCXM) was introduced into clinical practise by Garavoy et al. (13). The principle of the test involves incubating donor cells with recipient serum and then adding a fluorescein-labeled second anti-human immunoglobulin antibody that binds to patient antibody bound to the donor cells. The test is read on a flow cytometer, and the degree of positivity is expressed as a channel shift. The main advantage of the FCXM is its sensitivity for antibody detection over the conventional CDC crossmatch (13, 14). In cases where the second antibody is antihuman IgG, it is not possible to discriminate between complement binding (CB) and non-complement binding (NCB) HLA antibodies. However, if that additional information is required, it is possible to use second antibodies to the IgG isotypes and also IgM (15, 16). It is also possible to detect antibodies to both class I and class II HLA antibodies by using markers to differentiate T and B lymphocytes (17, 18).

With the advent of solid phase and in particular bead technology, and the interpretation of weak antibodies detected by those methods the flow crossmatch is used in many centers to assist in clinical decision making. For example, if the flow crossmatch is positive in the case where a weak HLA antibody is detected by the bead assay (19), a decision may be made to invoke a desensitization protocol or to not proceed with the proposed transplant depending on the patient's transplant and sensitization history. Alternatively, a weak HLA antibody detected by the bead assay in the presence of a negative flow crossmatch may result in the transplant proceeding, but again the patient's immunological history would be a part of the decision making in such a case.

#### Solid Phase Antibody Detection Assays Enzyme-Linked Immunosorbent Assay

The enzyme-linked immunosorbent assay (ELISA) was initially used in the HLA field for detecting levels of HLA both cell bound and free but was adapted for detection of HLA antibodies in serum in 1995 (20).

In the modified assay HLA glycoproteins are immune-precipitated usually from EBV transformed cell lines, and immobilized in the wells of microtiter trays. Sera to be tested are added to the wells and antibodies specific for the HLA molecules bind to the relevant epitope. After washing an anti-human IgG labeled with a reporter molecule such as alkaline phosphatase is then added which binds to the primary anti-HLA antibody molecule. After repeated washing to remove any unbound secondary antibody, a substrate is added which is dephosphorylated by the alkaline phosphatase resulting in a color change.

Two levels of testing are achievable using the ELISA technique. One involves the use of a pool of different class I and class II molecules, which essentially gives a positive or negative result, and the second utilizes HLA molecules derived from single individuals which can be used to determine antibody specificity.

The ELISA technique is more sensitive than CDC in detecting HLA antibodies (21, 22) but has the potential drawback of not distinguishing between complement-fixing and non-complement-fixing antibodies. This assay however has been used as a very effective method for detecting pre- and postsensitization in solid organ transplants (23–25) but has been somewhat superseded by the introduction of fluorescently labeled beads to which HLA molecules have been attached.

#### Luminex**®** Bead Technology

The introduction of fluorescently labeled beads revolutionized HLA antibody testing during the 1990s. Commercial kits are available (One Lambda, Immucor) which consist of beads impregnated with differing ratios of two fluorochromes resulting in a unique signal for each bead and which have one or several types of HLA molecules attached.

The assay involves first the incubation of a patient's serum with the beads. If the patient has HLA antibodies the serum will react with the bead expressing the appropriate HLA molecule. After washing, the beads are incubated with a secondary antibody, usually with a phycoerythrin (PE)-labeled anti-human IgG (**Figures 1** and **2**).

Three levels of testing are possible depending on requirements. The first level provides a positive/negative result with respect to a patient's antibody status. In this instance, the beads are bound with a large number of class 1 or class 2 molecules derived from lymphoblastoid cell lines. Beads used in second level testing are bound with molecules derived from a single cell line and hence express two HLA molecules for each of the HLA loci (HLA-A, -B, -C for class I and HLA-DR, -DQ and -DP for class II). This testing is essentially analogous to testing with a panel of cells, and therefore, the result can be expressed as a PRA percentage. The third level of testing involves the use of beads bound with single HLA molecules produced by recombinant technology, so called single antigen beads (SAB). These beads provide a real advantage of this technology as complex mixtures of antibodies can be characterized and HLA specificities accurately determined. This technology is now considered essential for the pretransplant testing of sensitized patients.

There are two common methods for the readout. The first method involves conventional flow cytometry and measuring the channel shift associated with antibody binding. The second which has become the most popular approach is the use of the Luminex® fluorocytometer which utilizes two lasers, one of which excites the fluorochrome in the bead and the other laser excites the PE bound to the detection antibody (**Figure 3**). The first readout therefore identifies the unique signal of the bead and hence the specificity of the bound HLA molecule, while the second readout indicates whether or not antibody is bound to the specific HLA molecule.

The degree of fluorescence is expressed as a mean fluorescence intensity (MFI), which is normalized by taking into account the degree of fluorescence observed with an antibody negative serum and with beads to which no HLA molecule is attached. A positive control consists of beads bound with PE-labeled human IgG.

FIGURE 1 | The figure represents the principles underlying the Luminex bead assay. Each bead has one or more different types of human leukocyte antigen (HLA) molecules attached depending on the level of testing being performed. If the test serum contains an HLA antibody it will bind to the appropriate HLA molecule. This binding can be detected by the use of a second phycoerythrin (PE)-labeled anti-human IgG. Each bead gives a specific signal when excited by one of the lasers built into the Luminex instrument due to the unique intensity of fluorophore embedded in the bead. A second laser detects the fluorescent excitation produced by the PE on the second antibody. The combination of the two signals indicates first the presence (PE fluorescence) and second the specificity (bead fluorescence) of the HLA antibody in the test serum.

FIGURE 3 | The top panel shows the Luminex instrument. There are two lasers in the Luminex instrument (bottom panel). The red laser excites the fluorophore in the bead which provides a unique signal thereby identifying the HLA molecule attached. The green laser excites the phycoerythrin bound to the second anti-human IgG antibody indicating IgG antibody in the test serum has bound to the appropriate HLA molecule attached to the bead. (Modified from a figure provided by Serologicals Corporation.)

## SUMMARY COMPARISON OF TECHNIQUES

The advantage of the CDC assay is that it is a functional test involving antibody containing serum and cells. As a crossmatch test it has proved invaluable over the years as a method of avoiding hyperacute rejection due to the presence of HLA-DSA in the recipient (3, 4). As an assay for screening patients for HLA antibodies it has drawbacks. First, it lacks the sensitivity of the other assays described, and second, the assignment of positive and negative reactions can be compromised by viability of the cells used. It also detects both IgG and IgM HLA antibodies in addition to autoantibodies and non-HLA antibodies against other cell surface determinants which have no relevance in organ transplantation.

In the context of organ transplantation, however, it does have the advantage of only detecting CB antibodies. The rationale for replacing this assay with the solid phase assays was driven primarily by the sensitivity issue and the realization that HLA antibodies positive by the solid phase assays but negative by CDC in some cases were clinically relevant (24, 26–29).

Before the introduction of the solid phase assays, the cell-based flow cytometry assay was introduced into clinical practice as a means of providing a more sensitive assay for detection of recipient presensitization to donor-specific HLA. The flow crossmatch however was not amenable to rapid turnaround times and therefore was used primarily in the living related and living unrelated donor situation rather than for cadaveric donors.

The issue of whether the flow assay was as sensitive as the solid phase assays was the subject of debate for some time but the general consensus is that the bead assays are the most sensitive assay for detecting HLA antibodies albeit with their own unique problems (30).

With respect to the two main solid phase assays the fluorescent bead assay has become the gold standard for HLA antibody detection and is now used in most transplant testing laboratories. The remainder of the review will concentrate on the advantages of this technique, and the challenges facing both laboratory workers and transplant clinicians in interpreting the data generated by this assay.

## ADVANTAGES OF THE LUMINEX**®** BEAD ASSAY

The Luminex® bead assay is a sensitive method for detection of HLA antibodies and represents the current highpoint in the evolution of HLA antibody detection assays. The additional sensitivity provided by this method has enabled the detection of HLA antibodies in potential transplant patients which are not detectable by other means, particularly CDC (24, 26–29). This increased sensitivity has enabled improvement in the success rate of retransplant patients due to the detection of HLA presensitization as a result of previous grafts and the subsequent avoidance of the relevant HLA specificities on second grafts particularly for DP specificities that are not detected by other methods (31).

The development of SAB has enabled the dissection and specificity determination of complex mixtures of HLA antibodies which is not possible with other techniques. This fine level discrimination coupled with the Matchmaker program (32) has enabled the description of epitope sequences to which HLA antibodies are directed (33–35). Armed with this information consideration of sequences of all alleles regardless of whether or not they are represented on the bead panel allows the identification of all HLA alleles to which a patient is immunized.

Obtaining HLA allele information on potential transplant recipients has permitted the identification of antibodies to alleles within the same antigen group. For example, an A\*0301 antibody identified in an A\*0302 renal transplant patient (36) and an A\*2402 antibody in an A\*2403 patient (37). The only coding sequence differences between A\*2402 and A\*2403 are located at positions 166 and 167 which are the unique substitutions within the epitope recognized by the A\*2403 patient. Historically, the presence of A\*2403 in a donor would have been considered an antigen match for an A\*2402 patient yet it clearly represents a potential immunizing situation.

Identification of antibodies to DQA1, DQB1, DRB3, -4, -5, and DPB1 which is not possible using other antibody screening assays has been enabled by the use of beads containing these molecules. As a result it has become evident that antibodies to DQ and DP (38–42) in addition to DR coexist in organ transplant recipients and have been implicated in negative graft outcomes.

## INTERPRETIVE CHALLENGES ASSOCIATED WITH THE LUMINEX**®** BEAD ASSAY

Many of the challenges in interpretation are described in a 2013 report of Consensus Guidelines by an expert Committee under the guidance of The Transplantation Society (43). In addition, reviews have appeared subsequently, which have further contributed to this topic based on more recent data (44, 45). Much of the data had been generated in renal transplantation but the technical issues apply equally to other forms of organ transplantation. The following outlines some of the major issues which require consideration when interpreting bead assay data.

## Sensitivity

Although the bead assay represents the most sensitive method for HLA antibody detection one of the main challenges facing clinicians and laboratory scientists is the interpretation of positive results in the context of a negative CDC crossmatch and/or a negative flow crossmatch, and no indication of presensitization by any other screening technique. The question of the clinical relevance of these HLA antibodies in rejection has been reported in renal, heart, and liver transplantation with mixed results (46–52). Many factors impact on the clinical relevance of these detectable low-level antibodies, one being the MFI cut off for positivity used by the reporting center.

# Mean Fluorescence Intensity

There is no recommended "cut off " value for MFI positivity. Most laboratories set their "cut off " level for positivity based on levels obtained with relevant controls and also on experience gained from clinical results obtained. A useful approach, particularly for multiparous females or previously grafted patients, is to consider each patient on an individual basis. For example, if a previously grafted patient has an MFI level for a particular HLA specificity to which they were exposed on the first graft and the MFI is above the negative values but below the "cut off " level established in the laboratory, this result should be treated with caution. It may indicate a state of presensitization with very low levels of antibody, the production of which can be reactivated on repeat exposure with a second graft bearing that antigen. Such a result may be interpreted differently in a patient with no history of potential HLA preimmunization.

Other factors such as the variable amount of target HLA present on the bead which can be locus and allele specific (53, 54) can result in variation of the MFI obtained. In the absence of an agreed standard for the performance of the Luminex® bead assay, it is incumbent on each testing laboratory to establish their own MFI "cut off " levels in consultation with their clinical colleagues.

# Antibodies to Denatured HLA

The SAB are coated with HLA molecules produced by recombinant technology while the screening beads are coated with HLA molecules immune-precipitated from cell lines. As a result, the SAB express denatured molecules in combination with native molecules. The denatured molecules can express cryptic epitopes not normally accessible by antibody molecules, and it is not possible to distinguish between these two types of antibodies. It would appear intuitive that since the antibody does not have access to the cryptic epitope that these antibodies will not be clinically relevant. However, a need existed for a means of distinguishing between antibodies to denatured and native epitopes. One manufacturer responded by introducing ibeads which are SAB expressing largely native HLA molecules. These beads however were removed from the market in 2014 and the manufacturer recommends as an alternative using an acid wash procedure.

Antibodies to these exposed cryptic epitopes on denatured molecules have been detected in individuals including nontransfused males (55, 56). Studies comparing antibodies to both denatured and native epitopes have demonstrated that the antibodies to denatured epitopes have no clinical impact in renal or heart transplantation (57, 58). Why antibodies to denatured epitopes are found in individuals, particularly non-transfused normal males, is a subject of debate. The concept of cross reactivity with environmental agents such as pathogens or ingested food has been suggested (56).

# Complement-Fixing and Non-Complement-Fixing HLA Antibodies

Unlike the CDC assay which by definition only detects CB HLA antibodies, the bead assay is designed to detect both CB and NCB antibodies. This created debate concerning the NCB antibodies detected by the bead assay, and the concern that many patients were being denied a transplant on the basis of donorspecific NCB antibodies, the clinical significance of which was not established.

Several modifications have been made to the assay to distinguish between CB and NCB HLA antibodies. Using anti-IgG2 and anti-IgG 4 antibodies to detect NCB antibodies Arnold et al. (59) were able to show that up to 40% of re-transplant patients on the waiting list had either HLA class 1 or II NCB antibodies. Wahrmann et al. (60) modified the flow-based bead assay by adding normal serum as a source of complement and anti-C4d as a second antibody and found similar incidence results to Arnold (61).

Modification to the Luminex® method of detection was first described by Chin and colleagues (62). Their approach involved heating the test serum to denature complement and then to add purified human C1q to the serum prior to incubation with the beads. CB antibody binds the C1q and then is detected using a secondary PE-labeled anti-human C1q antibody. This is now the method most commonly used by testing laboratories to distinguish between CB and NCB HLA antibodies. A commercial C1q kit is now available which can detect CB antibodies using either beads in the Luminex system or cells, or for use with cell-based flow cytometry.

The historical association of CDC positive crossmatches with hyperacute or acute rejection led many to believe that CB antibodies detected by the C1q assay would be associated with rejection while NCB antibodies would not. The reality however is that the associations are not so clear cut. Recent studies examining the clinical associations of antibody-mediated rejection with C1q CB and NCB HLA antibodies have yielded some interesting observations.

Calp-Inal et al. (63) showed that the incidence of both acute and chronic rejection was increased in those with CB DSA HLA antibodies pretransplant compared with patients whose DSA were NCB antibodies. Guidicelli et al. (64) demonstrated that while *de novo* CB HLA antibodies were associated with rejection shortly after their appearance NCB antibodies were also associated with rejection in the long term. Piazza et al. (65) also demonstrated a strong association of CB antibodies detected in the C1q assay with inferior graft survival and also demonstrated that the incidence of CB antibodies was increased among those patients with HLA class II antibodies, particularly DQ.

By contrast, Thammanichanond et al. (66) were unable to show a significant effect of CB antibodies with rejection, albeit it in a relatively small cohort of patients. They did however show that the CB antibodies had higher levels of MFI than NCB antibodies.

Likewise, Taylor et al. (67) claim the interpretation of the C1q assay is confounded by the level of antibody, the amount of denatured HLA on the beads and the interference of complement. They further question the justification of its use given the uncertainty in interpretation and the additional cost involved.

There are points to be made with respect to these studies. First, when pretransplant sensitization is involved, in the overwhelming majority of cases the CDC donor crossmatch is negative and therefore lower strength CB antibodies are being selected, which will impact on the overall clinical impact. Second, it is known that CB IgM antibodies which are not detected in the conventional SAB assay can convert to IgG3 CB antibodies posttransplant and are detrimental to the graft (68), which can have a confounding effect on the data analysis when pretransplant antibodies are analyzed. Finally, it appears that NCB antibodies may impact to a degree on graft survival at least in the long term (49, 64).

Recently, a C3d assay has been described (69), similar in principle to the C1q assay, which measures C3d deposition by the addition of human serum to the single bead antigen/antibody complex, followed by the addition of an anti-C3D antibody labeled with PE. Sicard et al. (69) were able to demonstrate in a group of renal transplant patients tested at the time of diagnosis for rejection, patients testing positive for C3d had a higher risk of graft failure. Interestingly, the C1q assay failed to reach statistical significance as a predictor of graft failure.

Comoli et al. (70) in a recent paper presented results of posttransplant testing monitoring for the appearance of *de novo* donor-specific antibodies. Positivity in the C3d assay did not predict graft rejection at the first appearance of *de novo* DSA but at the time of rejection there was a strong correlation. They also demonstrated that conversion within a single antibody from NCB to CB, as demonstrated by C3d positivity, was associated with an increasing MFI. The apparent greater association of rejection with C3d than with C1q may be a reflection of the stage of the complement cascade at which these assays are focused. As indicated by Comoli et al. (70), the presence of C1q does not predict whether the complement cascade will proceed, or just result in C4 deposition on the cell surface. The downstream production of C3d may more accurately predict complete complement activation.

#### The Prozone Effect

One of the technical challenges of using SAB for HLA antibody detection is the prozone effect whereby a diluted serum gives a higher MFI than the undiluted serum, suggesting an inhibitory effect which can be abrogated by dilution. One explanation for the inhibitory effect is the presence of IgM antibody of the same HLA specificity blocking the binding of the IgG isotype (71). Since IgM antibodies tend to be a lower titer than IgG the dilution effect was consistent with this interpretation. However, recent research suggests that the effect is due to the inhibitory effect of the C3 component of complement, which is produced as a breakdown product of C1. C3 binds to the beads and inhibits the binding of IgG antibody present in the test serum (72). This problem can be overcome by pre-heating of the test serum to destroy any complement activity or by the addition of ethylenediaminetetraacetic acid (EDTA) to the wash buffer (73) or by the use of dithiothreitol (71) which is also used to eliminate IgM antibodies by disruption of disulfide bonds. The possible confounding effect of prozone should be always considered when interpreting results obtained from using variations of the SAB assay for detection of complement-fixing antibodies.

#### THE DONOR-SPECIFIC LUMINEX CROSSMATCH

In 2008, Billen et al. (74) reported on the use of commercially available beads for donor-specific crossmatching. The beads are coated with one of two mouse antibodies with specificity for a non-polymorphic sequence on either the class 1 or class 2 molecules. Using lysates of donor cells the beads are able to capture the class 1 and class 2 molecules of the donor which can then be reacted with recipient sera and the bound antibody, if present, labeled with a fluorescently labeled second antibody and read as per the conventional bead assay. Billen et al. compared the results obtained in a group of renal patients with the cell-based FCXM results. They demonstrated a sensitivity using the bead crossmatch of 89% for class 1 and 68% for class 2. Interestingly, they failed to detect antibodies to HLA-DQ and -DP antigens by the bead crossmatch which compromised the value of the LUXM as an alternative B cell crossmatch technique.

Billen et al. (75) further reported on a group of 165 CDC crossmatch negative patients, 32 of whom had bead positive crossmatches. There was no difference in acute rejection free survival when the CDC-bead + crossmatch group were compared with the CDC-bead − crossmatch group. However, the group of patients with bead positive crossmatches due to class 1 antibodies had an inferior long-term 5 years survival (41% compared with 70% for the crossmatch negative group). Positivity for class 2 antibodies had no effect.

Guillaume et al. (76) demonstrated that the LUXM can detect class 1 antibodies with an MFI as low as 2,300 in the SAB technique and 1,300 for class 2. They confirmed the failure to detect HLA-DP antibodies and in addition reported on the failure to detect HLA-C.

Recently, Chaidaroglou et al. (77) reported on a comparison of SAB, FCXM, and LUXM for detection of DSA in a group of heart transplant recipients. They found that there was good agreement between SAB and FCXM but not between LUXM and the other two techniques. They questioned the value of LUXM as a technique for prediction or monitoring.

It would seem based on published data to date that the LUXM cannot be recommended as a stand-alone method for organ allocation.

#### THE VIRTUAL CROSSMATCH

The introduction of solid phase assays and the realization that there were a number of patients whose antibodies were detectable by these methods but were negative by the CDC crossmatch cast some doubt on the complete reliance on the CDC crossmatch as a final test of recipient/donor compatibility. Since antibodies with MFI values between 10,000 and 20,000 are required to obtain positive T cell CDC crossmatches in approximately 90% of cases (78), there are clearly some cases where clinically relevant HLA antibodies which are not detected by the conventional crossmatch. The use of specific and sensitive methods for antibody detection and in particular the HLA SAB allowed for the first time a complete picture of the HLA immunization status of individual patients. From this the concept of a "virtual crossmatch" (VXM) was established (79). The VXM takes into account the HLA antibody profile of a patient and predicts which donors will be crossmatch negative. This approach has been used successfully in renal transplantation. Johnson et al. (80) have reported on a large cohort of patients where the final decision to transplant was based on SAB results rather the FCXM result. When analyzed for clinical outcome based on whether the FCXM was positive or negative, despite the fact the FCXM positive group were more "at risk" than the FCXM negative group, the transplant outcomes were comparable, justifying the use of the VXM in preference to the results obtained by FCXM.

Eby et al. (81) have demonstrated the value of the VXM in pancreas transplants as part of the United Network of Organ Sharing in the USA. Pancreata imported from Networks 3 and 4 and transplanted on the basis of VXM had a cold ischemia 5 h shorter than the cases where a FCXM was performed prior to transplant without any compromise in rejection or graft survival incidences.

More centers are expected to rely on the VXM as a prospective guide to the suitability of transplantation as data on the reliability of this procedure is accumulated.

#### USE OF THE ANTIGEN BEAD ASSAY – LESSONS LEARNED

The introduction of solid phase HLA antibody detection methods and in particular the bead assays have revolutionized the clinical management of sensitized patients. However, the introduction of this new technology has posed questions concerning the interpretation of generated data which still require resolution.

The "cut off " MFI values used for assigning positivity vary greatly between laboratories. How much this is due to technical variation and how much is based on correlation with clinical experiences of the local transplant center is difficult to ascertain. Every testing laboratory must determine based on the local performance of the assay and from clinical experience a "cut off " that reflects the level at which antibodies are deemed to be clinically relevant. However in this context, it is imperative to consider the immunological history of the patient. Borderline values or values obtained with antibodies below the "cut off " may be reflective of an increased risk of rejection in patients who have been previously exposed to the particular antigen to which the antibody is directed either by pregnancy or previous grafts. One group of patients of particular interest are those who have undergone desensitization protocols. "Cut off " values therefore should be a guide and not rigidly enforced without careful consideration of the patients' histories.

The interpretation of HLA antibody results obtained with CB fixing assays requires careful interpretation. The prozone effect needs to be considered in patients who are known to be sensitized but test negative in the CB antibody assays. These patients should in addition be tested at a dilution or treated with EDTA or DTT prior to testing. HLA antibodies which test negative with the CB binding assays should not automatically be dismissed as clinically irrelevant. Although the data suggest in renal transplantation that NCB HLA antibodies are not as damaging to the graft as CB HLA antibodies, and tend to have lower MFI values, there are data which indicate that they do have a lower but nevertheless significant association with rejection. In the absence of convincing clinical data for NCB HLA antibodies in other solid organ transplants, they should also be treated with caution.

The relationship of positive bead assay antibody results with other assays is an important aspect of interpretation. There is universal agreement that an HLA antibody detectable by the bead assay which results in a positive CDC crossmatch is a contraindication to renal transplantation. However, a negative CDC crossmatch in such a situation is less clear cut. The MFI cut off for such a scenario is of the order of 10,000 below which the CDC crossmatch may be negative, but this may vary from laboratory to laboratory based on the sensitivity of the CDC crossmatch and the performance of the SAB under local conditions. Some centers have reported an increased risk of rejection in such patients, others have observed no effect. However, group analyses of such patient groups can mask individual patients whose negative outcome has been influenced by bead + CDC negative antibodies. Some centers recommend the use of a flow crossmatch in such patients. A negative flow and CDC crossmatch may be an indicator that transplantation can proceed. In most cases, this occurs in the presence of a low MFI by the SAB assay. However, the point must again be stressed that the interpretation of multiple assay results must occur in the context of the patient's history.

In some centers the CDC crossmatch has been replaced with the VXM. The inherent risk in this approach is that some patients will be denied a transplant in what would be a negative CDC situation and with HLA antibodies which may ultimately prove to be not graft damaging. The confounding factors such as prozone and denatured HLA on beads need careful consideration and analysis when relying on virtual crossmatching as the final pretransplant determinant of compatibility.

Published results using the LUXM crossmatch technique to date suggest that there are too many unresolved issues to recommend this technique as a stand-alone method for solid organ transplant allocation.

#### FUTURE DIRECTIONS

The introduction of solid phase assays ushered in a new era in antibody detection both in pre- and posttransplant patients. However, the question of B cell presensitization in patients whose primary antigenic stimulus was years previously and no longer have detectable circulating antibodies still represents a challenge. This problem was referred to briefly in the section on MFI where levels of antibody below the accepted level for positivity in previously grafted patients or multiparous females should be flagged as a potential problem as it may indicate a state of presensitization. Knowing the HLA genotype of previous transplant donors or the biological father of the multiparous patients' children can be useful in this regard.

Cardiac grafts in multiparous females with a negative CDC crossmatch have a higher incidence of rejection if the donor shares an HLA with the father of the patient's children (82) even when the primary immunizing pregnancy was up to 30 years previously, demonstrating the long-term effect of memory T and B cells. A comparable study considering HLA epitopes rather than broad antigens has not been reported.

More recently, Mulder et al. (83) used HLA tetramers to study the peptide dependency of HLA antibodies. This technology was utilized by Zachary et al. (84) to investigate the incidence of HLA class I memory B cells in patients with a history of HLA antibodies, but shown to be HLA antibody negative when testing current sera samples. Using tetramers labeled with PE and a labeled CD19 antibody to identify B cells, flow cytometry identified a percentage of cells which bound to the HLA tetramers. The incidence of bound tetramers was significantly greater in previously HLA immunized compared with non-immunized individuals indicating the presence of memory B cells.

A recent report using the ELISPOT assay and HLA class II biotinylated molecules has described the detection of HLA class II-specific memory cells (85) indicating this assay is a useful tool for identifying presensitization in the absence of circulating antibodies (86).

In addition to previously grafted patients and multiparous patients, the use of this technology has potential in monitoring patients who have undergone desensitization protocols prior to transplantation. Depending on the type of desensitization procedure used, it is useful to establish if memory B cells can be detected in patients whose circulating HLA antibody is no longer detected after treatment.

# CONCLUSION

The introduction of solid phase and in particular bead-based assays, for detection of HLA antibodies has revolutionized clinical management of organ transplant patients. For the first time, laboratory scientists and clinicians are in a position to fully reveal a patients immunological status. This technological breakthrough coupled with HLA sequencing data, which permits the identification of epitopes to which HLA antibodies are directed provides a unique opportunity to maximize the transplant success rate. This new found enthusiasm however is tempered by the fact that there are still areas of both technical and clinical contention which require resolution, such as the role of NCB HLA antibodies and the detection of antibodies to denatured HLA antibodies and their role if any in graft rejection. With this rapid rate of evolution of HLA antibody testing technology, it is imperative that laboratory-based scientists and clinicians communicate on an individual patient basis. Having regard to the immunological history of the patient when interpreting HLA

#### REFERENCES

antibody results is critical in maximizing the positive clinical impact of this technology.

#### AUTHOR CONTRIBUTIONS

The sole author was totally responsible for the design and writing of the review.

#### ACKNOWLEDGMENTS

Australian governments fund the Australian Red Cross Blood Service for the provision of blood, blood products, and services to the Australian community.

*and Causing Acute Humoral Rejection and Kidney Graft Dysfunction*. Los Angeles: Clinical Transplants Pub, Terasaki Foundation Laboratory (2006). p. 379–83.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Tait. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Significance of Anti-HLA Antibodies on Adult and Pediatric Heart Allograft Outcomes

#### *Massimo Mangiola1 \*, Marilyn Marrari1 , Brian Feingold2 and Adriana Zeevi1 \**

*1Division of Transplant Pathology, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA, 2Pediatric Cardiology, The Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA*

#### *Edited by:*

*Narinder K. Mehra, All India Institute of Medical Sciences, India*

#### *Reviewed by:*

*Stanislaw Stepkowski, University of Toledo, USA Reem Al-Daccak, French Institute of Health and Medical Research, France*

#### *\*Correspondence:*

*Massimo Mangiola<EMAIL_ADDRESS>Adriana Zeevi<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

*Received: 13 September 2016 Accepted: 03 January 2017 Published: 27 January 2017*

#### *Citation:*

*Mangiola M, Marrari M, Feingold B and Zeevi A (2017) Significance of Anti-HLA Antibodies on Adult and Pediatric Heart Allograft Outcomes. Front. Immunol. 8:4. doi: 10.3389/fimmu.2017.00004*

As methods for human leukocyte antigens (HLA) antibody detection have evolved and newer solid phase assays are much more sensitive, the last 15 years has seen a renewed focus on the importance of HLA antibodies in solid organ transplant rejection. However, there is still much controversy regarding the clinical significance of antibody level as depicted by the mean fluorescence intensity of a patient's neat serum. Emerging techniques, including those that identify antibody level and function, show promise for the detection of individuals at risk of allograft rejection, determination of the effectiveness of desensitization prior to transplant, and for monitoring treatment of rejection. Here, we review current publications regarding the relevance of donor-specific HLA antibodies (DSA) in adult and pediatric heart transplantation (HT) with graft survival, development of antibody-mediated rejection and cardiac allograft vasculopathy (CAV). The negative impact of DSA on patient and allograft survival is evident in adult and pediatric HT recipients. Many questions remain regarding the most appropriate frequency of assessment of pre- and posttransplant DSA as well as the phenotype of DSA memory vs. true *de novo* antibody using large multicenter adult and pediatric cohorts and stateof-the-art methodologies for DSA detection and characterization.

Keywords: AMR, heart transplantation, donor-specific antibodies, allograft vasculopathy, graft failure

#### INTRODUCTION

Heart transplantation (HT) has become an accepted therapy for adult and pediatric patients with end-stage heart failure. Despite improved immunosuppression regimens, rejection remains the most common cause of death in the first 5 years after HT. Both cellular and humoral immunemediated processes that can damage the allograft are primarily directed against human leukocyte antigens (HLA). Antibodies against HLA can be found in patients prior to transplantation after exposure to foreign HLA through blood transfusion, pregnancy, previous transplant, and use of homograft tissue during surgery for some congenital heart defects. Ventricular assist devices (VAD) have also been implicated in the development of HLA antibodies, termed allosensitization. Exposure to donor HLA after HT may also induce *de novo* production of donor-specific HLA antibodies (DSA). The impact of circulating HLA antibodies on heart allografts has been the focus of many investigations and reviews. The introduction of solid phase assays (SPA) based on the luminex single antigen bead assay (SAB) has improved the sensitivity and specificity of HLA antibody detection; however, it also introduced new challenges for assay interpretation and determining its clinical relevance (1).

Identification of DSA enables the clinician to make informed decisions regarding acceptance of the organ and the choice of immunosuppression (2). Presence of DSA is not always considered a contraindication but rather a risk factor for organ transplantation success (3). Optimizing transplantation of allosensitized candidates is challenging and program specific. The main challenge with the new SPA technology is decision-making regarding donor organ acceptance based solely on antibody strength determined by mean fluorescence intensity (MFI) (2–5). The threshold for accepting a donor for a sensitized patient may vary depending on the patient's clinical status, antibody level, and protocols available for antibody removal therapy. Considering the SPA modification to detect complement-fixing antibodies (C1q-SAB) has reduced the estimated incompatible donor pool in highly sensitized patients (6). Optimizing transplantation of allosensitized candidates using SAB and C1q-SAB methodology to prioritize the assignment of unacceptable antigens has allowed transplantation of highly allosensitized patients across the DSA barrier with survival rates comparable to DSA− heart transplant recipients (5).

Titration of sera prior to SAB testing has emerged as a more accurate way to assess the true level of DSA as compared to MFI value of undiluted sera (7). Furthermore, titration studies provide better estimates of responsiveness to antibody removal therapies (8).

Recognition that some preformed antibodies are against denatured HLA antigens with very little clinical relevance may also impact the search for an acceptable donor (4, 9). The assignment of unacceptable antigens has been greatly improved also by incorporating patterns of epitope reactivity and history of sensitizing events. Recognizing the limitations and advantages of current available methods for antibody determination, quantitation and function has facilitated the introduction of the virtual crossmatch (VXM) in thoracic transplantation. Previously, the need for prospective crossmatch (XM) in sensitized patients was associated with longer waitlist duration and increased mortality (10). Although VXM is widely used for organ allocation, its validity highly depends on how accurate and current is the information on patient sensitization events and comprehensive DNA-based HLA typing of prospective donors as antibodies can be made against every possible polymorphic HLA target antigen (2–5).

#### RELEVANCE OF DSA ON OUTCOMES

In this report, we focus on a short review of the current stateof-the-art regarding the role of DSA in adult and pediatric HT as determined by the following outcome measures: graft survival (GS), development of antibody-mediated rejection (AMR), and cardiac allograft vasculopathy (CAV) (**Tables 1** and **2**). Although we limit this review to the last 6 years, the retrospective nature of some studies may influence the relevance of DSA on clinical outcomes due to the use of less sensitive testing methods. Furthermore, we considered separately the role of DSA on adult and pediatric clinical outcomes to highlight potential similarities and differences in the two cohorts.

## ADULT HT

#### Graft Survival

The prevalence of allosensitization in heart transplant candidates increased with the introduction of SPA for screening for HLA antibodies (11) (**Table 1**). Nevertheless, the risk for poor GS has remained a significant finding even in the more sensitive SPA testing era (11, 12). The presence of non-cytotoxic HLA antibodies identified by SAB was associated with high risk of death, early graft failure, and late cellular- and antibody-mediated rejection; these findings underscore the need for using sensitive Luminex platform SPA to accurately determine the presence of circulating HLA antibodies (12). Detection of Class I DSA pretransplant was a predictor of short-term but not long-term survival as compared to non-DSA (13). In this study limiting the testing on pretransplantation, the authors could not identify the impact of persistent vs. transient DSA and of *de novo* DSA on clinical outcomes.

*De novo* antibody production and its role in cardiac allograft survival has been described in several studies (14–16). In a retrospective adult cohort, *de novo* DSA was associated with poor patient survival (HR = 3.198), while *de novo* and persistent DSA was worst (HR = 4.351) (14). Similarly, patients with persistent *de novo*, mostly Class II DQ-specific DSA, had worse survival (15). The 15-year survival was highest in patients who never developed DSA vs. those that developed DSA posttransplantation (70 vs. 47%), and patients with late *de novo* DSA appearing more than 1 year post transplantation had poorest survival (16). Thus, determining the presence of DSA pretransplant for risk assessment and monitoring for persistent and *de novo* DSA posttransplant provide the most comprehensive information for clinical management.

#### Antibody-Mediated Rejection

The challenges of AMR diagnosis post HT have been addressed by many single-center studies and consensus conferences. In the current era, diagnosis of this clinically important entity has been improved by standardized classification of histologic and immunologic changes in endomyocardial biopsies (EMB) (17) and by advances in the detection of HLA antibodies. Although not required for diagnosis of pathologic AMR (pAMR), the detection of HLA antibodies pre- and posttransplantation has been helpful for risk stratification for the development of AMR and for guiding treatment strategies (18). Patients with positive VXM defined in the presence of DSA >1,500 MFI by SAB had a higher incidence of AMR and cell-mediated rejection. Similar outcomes were observed with positive flow crossmatch (FXM) suggesting that SAB MFI >1,500 can be used as surrogate for FXM (19). Increased risk for a positive complement-dependent cytotoxicity (CDC) XM and early AMR was observed in patients with persistent C1q+ DSA (20). However, patients who had DSA but lost the C1q reactivity posttransplant did not develop early AMR, and the strength of neat sera on SAB did not predict C1q reactivity. In contrast, high titer DSA (>1:16) has been associated with

*assay; VLR, very late rejection; y, year(s); HLA, human leukocyte antigens.*

complement-fixing reactivity (7, 20) and has been used to determine unacceptable HLA antigens for sensitized candidates (5). DSA determination by SPA and elevated peak panel-reactive antibodies (PRA) were independent predictors of pAMR in an adult cohort of heart transplant recipients (21). In this study focusing on pretransplant samples, increasing numbers of DSAs and the mean cumulative MFI of DSAs were associated with risk of AMR, and the subset of C1q-reactive DSAs were less informative (21). Pathologic classification of AMR in 37 EMB correlated with circulating DSA and endothelial activation (22). The proportion of DSA+ EMB varied according to pAMR grade, and pAMR2 was associated with 100% DSA positivity (22). The clinical significance of DSA level as depicted only by MFI of neat serum is still controversial, and currently multiple approaches are proposed to capture the DSA level and function, including serum titration and complement-binding assays. A better concordance was observed between C1q+DSA and C4d immunofluorescence (IF)+ staining in EMB as compared with total IgG DSAs and C4d IF+ in EMB among 44 recipients (40 vs. 24%, *p* = 0.02) (23). A majority (82%) of patients with graft dysfunction had circulating C1q+ DSAs (23). However, not all patients with circulating C1q+DSA had C4d IF+ staining on EMB, suggesting that the presence of C1q+DSA may precede the development of pAMR or be due to the low sensitivity of C4d IF staining (23).

Prognosis after late AMR (defined as AMR >1 year posttransplant) was poor in 20 recipients despite aggressive treatment with immunosuppression, and fulminant CAV was a common condition (24). DSA was present in all tested patients (*n* = 19) with a median cumulative MFI at diagnosis >10,000; most of the patients had *de novo* DSA (24). Antibody-mediated injury and immune-mediated coronary arteriosclerosis were the causes of late graft failure in a recent study of 40 explanted heart allografts (25). AMR was observed in 47.5% failing heart allografts, including 40% of patients in whom unrecognized previous episodes of subclinical AMR occurred years before allograft loss. Among the 19 patients with AMR, 15 were tested for DSA, and 93% had circulating DSA at the time of allograft failure. The immunodominant DSA was Class II in 11/14 DSA+, and the median DSA MFI was 5,000 (25). In contrast, only 37% of patients without AMR features at the time of allograft failure had circulating DSA as compared to the AMR group (*p* < 0.001), and the median DSA MFI was 1,250 (25). In a retrospective cohort study spanning over 10 years, the timing of AMR (early vs. late) was associated with GS and CAV (26). Patients were tested at the time of biopsy for circulating DSA either by CDC (before 2010) or SAB (post 2010). Graft dysfunction was less frequent in early AMR, while late AMR with graft dysfunction showed rapid development of *de novo* CAV despite aggressive treatment and also increased risk of death (26).

#### Cardiac Vasculopathy

Cardiac allograft vasculopathy continues to remain a limiting factor in long-term survival of heart transplant recipients, and there is increasing evidence of the negative impact of circulating DSA on the development and severity of CAV. Patients with DSA had significantly higher rates and a shorter mean time to CAV and increased severity of CAV as compared to patients without DSA (27, 28). Patients with very late rejection and circulating DSA with evidence of intravascular macrophages had an increased risk of severe CAV as compared to patients without DSA (29).

# PEDIATRIC HT

## Graft Survival

Allosensitization and GS in pediatric HT recipients have been evaluated in large single- (30) and multicenter datasets (31, 32) (**Table 2**). Pediatric patients with PRA >10% had earlier-onset graft vasculopathy (30) and worse graft and patient survival than did patients with PRA <10% (31, 32). Elevated PRA was an independent risk factor for worse long-term GS (31). Furthermore, significant allosensitization (PRA >50%) at listing was associated with a more than twofold increased risk of death within the first transplant year (32). These large patient cohorts that were transplanted over a period of 18 years may have underestimated the rate of allosensitization because the methodology for PRA screening evolved from a less sensitive cell-based method to the more sensitive SPA. In addition, the SPA may have also increased the need for prospective XM due to an increased use of VXM (33).

In a more recent study patients with PRA >25% had significantly (*p*= 0.004) decreased survival compared to those with PRA <25% (34). In contrast, the outcome of allosensitized pediatric patients with PRA >10% who were desensitized was not different than non-sensitized recipients (35).

Assessments of GS in the presence of DSA show somewhat mixed findings, perhaps related to the duration of follow-up. Although short-term GS was not impacted by the presence of DSA in one pediatric study (36), the 5-year survival was significantly better in patients without DSA in another pediatric cohort (72 vs. 21%) (37). While uncommon, the presence of *de novo* DSA posttransplantation, especially toward Class II HLA, was associated with increased graft loss (38).

Multiple factors appear to play a role in development of *de novo* DSA in pediatric HT including prior sensitizing events, older age, African-American race, and donor death from gunshot wound (39). Knowledge of risk factors for the development of *de novo* DSA in pediatric recipients is likely to be important to guide the frequency of monitoring for HLA antibodies (39).

# Antibody-Mediated Rejection

Current understanding of AMR after HT is largely derived from adult studies. Using the Pediatric Heart Transplant Study database, the reported incidence of AMR was 11% (among 1,596 recipients), and patient and GS were lower for those with AMR (40). Risk factors associated with AMR included PRA >10% at HT, a positive CDC XM, and congenital heart disease, suggesting allosensitization related to the use of homografts (40).

The proportion of AMR-free patients was much higher among patients with only solid phase-detected DSA vs. those with CDCdetected DSA (41). Similarly, using the C1q assay, which detects only complement-fixing antibodies, the presence of C1q fixing DSA prior to or early after HT had a positive predictive value of 100%, while absence of C1q fixing DSA had a negative predictive value of 100% for AMR (42). In another analysis, the presence of circulating DSA had 93% sensitivity, 62% specificity, 24% positive predictive value, and 99% negative predictive value for biopsy diagnosis of AMR in pediatric recipients (43). In addition, higher levels of circulating DSA measured by MFI correlated with pAMR severity (43). The authors proposed that DSA monitoring provides a non-invasive tool to tailor the frequency of biopsy surveillance (43). Others have used an institution-specific MFI threshold value for DSA of >6,000 that strongly correlated with C4d deposition on EMB with high negative predictive value (97%) and specificity (95%) (44). Both studies emphasized the advantage of following DSA in asymptomatic pediatric patients, given the value of early detection of AMR (43, 44).

Similar to findings for renal transplantation (45), sensitized recipients with persistent posttransplant DSA with complementfixing ability appear to be at high risk for AMR (20, 42, 46).

#### Cardiac Vasculopathy

Overall, DSA+ patients (preformed or *de novo*) had significantly higher rates of CAV compared with DSA− patients. By 5 years, the rate of CAV-free survival was 25% for DSA− vs. 0% for DSA+ (37). Persistent DSA was associated with poor outcome and development of CAV (47).

# TREATMENT

Desensitization is aimed to increase the donor pool by either reducing or eliminating HLA allosensitization or by facilitating transplant by reducing the DSA burden. Desensitization treatment targets critical components of the humoral response to either achieve a negative crossmatch pretransplant or to reduce the impact of DSA in positive crossmatch transplants. At low titer, antibody reduction can be achieved with plasma exchange and IVIG. The use of B cell suppression agents (rituximab), plasma cell depletion agents (bortezomib), or inhibitors of complement activation (eculizumab) is usually limited to highly sensitized patients. The current literature in adults is not abundant, mostly observational, with small cohorts, short follow-up, and with inconsistent treatment methodologies (48–53). In 21 highly sensitized patients, the use of plasmapheresis (PP), IVIG, rituximab, and cyclophosphamide resulted in comparable long-term survival when compared to the low sensitized and unsensitized cohorts (53). A recent experience with bortezomib and PP showed that about 50% of the patients had a calculated PRA reduction and were transplanted with a negative crossmatch (48, 52). One year follow-up showed 100% survival and 74% freedom from rejection (48, 52). In a smaller cohort of patients transplanted across a positive crossmatch and treated with eculizumab and ATG, 1-year survival was 89%, and freedom from rejection was 75% (52). In patients treated for AMR, Class I HLA antibodies demonstrated a statistically significant response to bortezomib, whereas Class II responded poorly (51).

In pediatric HT, requiring a negative prospective crossmatch increases the waiting time and more importantly the waitlist mortality (10). Allosensitization is most significant among children with certain forms of congenital heart disease due to the use of homograft during prior surgeries. Also, blood transfusions and VAD use are common causes of allosensitization. Current literature for pediatric heart transplant desensitization is even more limited than in the adult cohort (54–58). Desensitization was carried out successfully with bortezomib and PP in a pediatric setting (54). Furthermore, in a single-center retrospective study in a large cohort of patients, all sensitized patients received PP or plasma exchange preoperatively. If the cytotoxic XM was positive, PP was continued. Patients with negative XM did not receive additional PP and IVIG posttransplant (58). Hemodynamically significant AMR occurred in 50% of patients transplanted across a positive XM vs. 2% of the XM-negative cohort (58). Additionally, incidence of serious infection was higher in patients transplanted across a positive crossmatch (58). Antibody depletion therapies were also used in management of AMR in pediatric patients. Decreased DSA MFI in 21 patients treated with PP correlated with good clinical outcome (55). In another small study, addition of bortezomib to PP and rituximab treatment resulted in a rapid decline in DSA and reversal of AMR without significant side effects (56).

## SUMMARY

The negative impact of DSA on patient and allograft survival is evident in adult and pediatric HT recipients. Allosensitization depicted by PRA >10% using cell based (prior era) or SPA (current era) is associated with poor outcome in both cohorts. Furthermore, similar risk factors were identified in adults and pediatric recipients for the development of posttransplant DSA including sensitizing events pretransplant, ECMO, need for mechanical support, non-compliance, and African-American race. In adults, but not in pediatrics, female gender (prior pregnancies) was also associated with a higher risk for development of *de novo* DSA. In children, exposure to homografts as part of surgical repair for some forms of congenital heart disease increase their risk for allosensitization and AMR.

Many questions remain regarding the most appropriate frequency of assessment of pre- and posttransplant DSA as well as the phenotype of DSA memory vs. true *de novo* antibody using large multicenter adult and pediatric cohorts and state-of-theart methodologies for DSA detection and characterization. The observation that early vs. late AMR in HT may have different prognosis and responses to treatment emphasizes the need to assess the risk of sensitization pretransplantation and to follow by routine monitoring of DSA posttransplant.

The ongoing multicenter clinical collaborative studies supported by National Institute of Health in adult and pediatric HT will hopefully provide answers to many remaining questions regarding the impact of preformed and *de novo* DSA on clinical outcomes and the efficacy of various modalities for desensitization and treatment of AMR.

# AUTHOR CONTRIBUTIONS

This is an invited review on impact of HLA donor-specific antibody in cardiac allograft outcome. All authors contributed equally to literature review, summary, and manuscript preparation.

# REFERENCES

heart transplant patients using current management strategies. *J Heart Lung Transplant* (2015) 34(2):175–81. doi:10.1016/j.healun.2014.09.027

*J Heart Lung Transplant* (2011) 30(12):1320–6. doi:10.1016/j.healun.2011. 08.009

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Mangiola, Marrari, Feingold and Zeevi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Unraveling the Role of Allo-Antibodies and Transplant Injury

*Yoshiko Matsuda and Minnie M. Sarwal\**

*Department of Surgery, University of California San Francisco, San Francisco, CA, USA*

Alloimmunity driving rejection in the context of solid organ transplantation can be grossly divided into mechanisms predominantly driven by either T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), though the co-existence of both types of rejections can be seen in a variable number of sampled grafts. Acute TCMR can generally be well controlled by the establishment of effective immunosuppression (1, 2). Acute ABMR is a low frequency finding in the current era of blood group and HLA donor/recipient matching and the avoidance of engraftment in the context of high-titer, preformed donor-specific antibodies. However, chronic ABMR remains a major complication resulting in the untimely loss of transplanted organs (3–10). The close relationship between donor-specific antibodies and ABMR has been revealed by the highly sensitive detection of human leukocyte antigen (HLA) antibodies (7, 11–15). Injury to transplanted organs by activation of humoral immune reaction in the context of HLA identical transplants and the absence of donor specific antibodies (17–24), strongly suggest the participation of non-HLA (nHLA) antibodies in ABMR (25). In this review, we discuss the genesis of ABMR in the context of HLA and nHLA antibodies and summarize strategies for ABMR management.

#### *Edited by:*

*Narinder K. Mehra, All India Institute of Medical Sciences, India*

#### *Reviewed by:*

*Rhonda Holdsworth, Australian Red Cross Blood Service, Australia Takuya Ueno, Harvard Medical School, USA*

> *\*Correspondence: Minnie M. Sarwal<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

> *Received: 03 June 2016 Accepted: 03 October 2016 Published: 21 October 2016*

#### *Citation:*

*Matsuda Y and Sarwal MM (2016) Unraveling the Role of Allo-Antibodies and Transplant Injury. Front. Immunol. 7:432. doi: 10.3389/fimmu.2016.00432*

Keywords: HLA antibody, donor-specific HLA antibody, non-HLA antibody, antibody-mediated rejection, humoral immune system, *in vitro* B cell assay

#### INTRODUCTION

Organ transplantation improves the quality of life of patients with terminal dysfunction of organs, such as the kidney and pancreas, and is the most effective life support treatment for patients with heart, lung, and liver failure.

Although short-term prognoses for transplanted organs have improved significantly, long-term prognosis after 5–10 years remains insufficient, and reportedly reflects injury from chronic, indolent injury from sub-clinical antibody-mediated rejection (ABMR) (3–5, 15). Acute ABMR is a declining problem in organ transplantation as donor/recipient matching has improved (7, 16) and early acute ABMR is seen usually only in the context of ABO incompatible organ transplants (17, 18), and transplantation in highly sensitized patients with preformed donor-specific HLA antibodies (DSAs). Accordingly, preformed DSA are more likely to be produced before transplantation with histories of complications, such as pregnancy, previous transplant, blood transfusion, and prior organ transplantation (7, 19, 20). Hyper acute rejection, which can occur in the presence of preformed DSA, can be controlled using recently developed desensitization therapies (7).

Rejection due to *de novo* DSAs remains a major cause of transplanted organ loss, in the context of sub-clinical, chronic ABMR (21–24). Moreover, ABMR has also been reported in the absence of DSAs, leading to the discovery of specific non-HLA (nHLA) antigens that activate humoral immune responses in the graft. Potentially, nHLA antibody-mediated humoral immune responses develop acutely and chronically following transplantation and these antibodies may influence prognoses by participating in the onset and sequelae of rejection (16–18, 25–33). Although graft rejection has been reported among patients with nHLA antigens, one of challenges has been the discovery of the identity of these novel nHLA antigens and to correlate their presence and titers with ensuing mechanisms of transplant rejection.

# MOLECULAR PATHOPHYSIOLOGY

During ABMR, antibodies for donor antigens are produced following activation of humoral immune responses, involving activated T cells and complement pathways.

As shown in **Figure 1**, naïve B cells differentiate into DSAspecific plasma cells (PCs) via germinal centers following exposure to antigens. This process involves initial uptake and surface presentation of donor antigens on antigen-presenting cells (APC) in response to an encounter of donor antigens, leading to activation of CD4+ effector T cells (34) and successive promotion of class-switching of naïve B cells and differentiation of memory B cells into PCs (35). Transmission of CD4+ effector T cell signals to B cells primarily involves association of major histocompatibility complex 1 (MHC-I) with T cell receptors (36). In addition, subordinate signaling pathways are activated by binding of CTLA4 (CD152), CD28, and CD40 ligand (CD40L) on T cell surfaces to the B7 (CD80/86) complex and CD40 on B-cell surfaces. Although CTLA-4 binds to B7, it reportedly downregulates T cell activity by binding to B7 with much greater affinity than CD28 (37–40). Intracellular CTLA-4 was closely related to the suppressor function of regulatory T cells (41–43) and reported the close relationship with autoimmune disease, including Graves' disease, type 1 diabetes mellitus (DM) (44–48).

CD28 is expressed on CD4+ effector T cells and naive T cells (47), and promotes interleukin (IL)-2 production from B cells following binding to B7 complexes (48), leading to sustained

naïve B cell differentiation into memory B cells (49). Conversely, CD40L mediates the class-switch of B cells in the germinal center by binding to CD40 expressing B cells (50) and support CD4+ effector T cells to help B cell differentiation (51, 52). Previous studies by Ettinger et al. (53) also showed that IL-21 induced PC phenotypes of human naïve and memory B cells following stimulation through B cell receptor (BCR) and CD40. Therefore, DSA-specific PCs developed and produced DSAs.

# THE ROLE OF COSTIMULATORY PATHWAY IN THE CLINICAL FIELD

CTLA-4Ig (immunoglobulin) binds to B7 and then suppresses the engagement of CD28. CTLA-4Ig can suppress the function of activated T cells through regulatory T cells, which may help suppress established chronic inflammatory disease (54).

In the field of transplantation, Belatacept, which links to the extracellular domain of CTLA-4, has been approved for the treatment of acute kidney rejection. In addition, an important problem in the field is to control antigen-specific memory B cells differentiation into PCs. The infusion of Belatacept might suppress DSAs development in a T-cell-independent manner because it has been reported that the infusion of CTLA-4Ig 1 week or more after transplantation could prevent DSAs development in a fully mismatched mouse cardiac transplant model but did not affect T-cell function (55). In addition, in the recent clinical BENEFIT trial of Belatacept induction by Vincenti et al. (56), there was a significant lower incidence of DSA development with Belatacept induction, when compared to the standard CNI arm, despite the higher incidence of acute rejection seen early with Belatacept induction.

The results might indicate that CTLA-4Ig could inhibit the growth and survival of DSA-specific memory B cells or PCs in a human model. About the other suppressive receptors related to CD28, PD-1 (programed death-1) has been reported to be expressed on the surface of T cells, and B and T lymphocyte attenuator (BTLA) has been reported to be expressed on the surface of both B and T cells, both of which have also been attracting attention as targets for treating autoimmune diseases and cancer (57).

In the field of autoimmune disease, the involvement of signaling through CD40–CD40L interaction in autoimmune diseases has been reported and dysregulation of CD40 may induce macrophage-mediated coronary artery disease (CAD); the blockade of CD40L may, thus, be an attractive therapeutic target to improve CAD (58). Recent studies have also implicated altered regulation of the CD40 axis and generation of pathogenic activating anti-CD40 antibody for the generation of podocyte injury in focal segmental glomerulosclerosis (FSGS) recurrence after kidney transplantation (59, 60). Further research is needed to better elucidate how the CD40 axis may help control other autoimmune diseases.

## HUMAN LEUKOCYTE ANTIGEN ANTIBODIES

Histocompatibility analyses using cross-match, human leukocyte antigen (HLA) typing, and antibody tests are widely performed prior to transplantation in many laboratories, and are an accepted approach for limiting organ rejection. Recent developments in laboratory procedures, survey equipment, and technologies have led to highly sensitive detection of HLA antibodies.

Therefore, we could detect a very small amount of HLA antibodies and determine these antibody specificities; trace quantities of HLA antibodies recently provided useful prognostic information for ABMR and transplanted organ outcome and a judgment of transplant evaluation (61–65).

Major histocompatibility complex 1 class 1 (HLA-A, -B, and -C) and MHC class 2 (HLA-DR, -DP, and -DQ) have been identified as HLA antigens, and HLA antibodies can be detected in sera using FlowPRA® Class I & II Screening Tests (One Lamda) to identify Class I or/and Class II HLA antibodies. In further analyses, positive cases should be identified using HLA LAB Screen HLA Class I or/and Class II single antigen beads (One Lambda) with Luminex technology, which determines antibody profiles against HLA Class I or Class II and indicates the presence or absence of DSAs.

# THE ROLE OF PREFORMED DSA IN THE PATHOGENICITY OF GRAFT INJURY

Donor-specific HLA antibodies that cause ABMR have been classified as those that are present before transplantation as well as those *de novo* that are produced after transplantation. Previous studies on kidney, heart, lung, and liver transplantation indicate that poor-prognosis is associated with the presence of DSAs before transplantation. We will next discuss the role of preformed DSAs in each organ transplant. With regard to kidney transplants, preformed DSAs have been recognized as one reason of hyper acute rejection. DSAs with high threshold MFI and DSAs with cross match-positive could predict ABMR onset after transplantation (7). With regard to pancreas transplants, we found a report describing that preformed DSAs did not affect graft prognosis (66) but DSAs could be detected from the sera with significantly higher probability than in recipients without a history of preformed DSAs after transplant. As a result, recipients sensitized by DSA before transplant had a history of DM more than 10 years after the transplant, so we should pay more attention to postoperative management, including blood sugar management (67).

With regard to liver, heart, and lung transplants, it is already known that preformed DSAs could affect graft outcome and patient mortality. In addition, preformed C1q binding DSAs have been reported to affect graft prognosis in liver and heart transplants and preformed DSAs with MFI ≥5000 and IgG3 DSAs could be risk factors for ABMR onset in liver transplant cases (19, 68, 69). Indeed, additional risk factors for ABMR in these patients include ABO incompatible and cross match-positive status, cases with a history of previous transplants, pregnancy, and blood transfusions (19, 70, 71). With regard to lung transplants, preformed DSAs have been reported to promote *de novo* DSA development early after transplant and patient survival (72–75). Therefore, these data on clinical correlations of DSA and rejection in different organ transplants suggests that improved screening and therapies, such as desensitization before transplant, may be of benefit across different types of solid organ transplants to limit subsequent postoperative complications (76).

# Mechanisms of Onset of ABMR by Preformed DSA

Antibody-mediated rejection caused by preformed DSAs manifests as hyper acute rejection immediately after transplantation, leading to failure of the transplanted organ within several hours. In these cases, DSAs immediately bind to all capillary endothelium surfaces in the transplanted organ, and concomitant complement activation leads to the formation of fibrin clots and acceleration of blood coagulation. Subsequently, rapid peripheral circulation incompetence causes necrosis of vascular walls, intense bleeding of the transplant, and necrosis in neighboring tissues. Finally, inflammatory cells, such as neutrophils, infiltrate capillary endothelial surfaces, and further undermine the transplant (6, 77).

#### Management of ABMR by Preformed DSA

Improvements in desensitization therapy have enabled management of high risk recipients, such as those with cross matchpositive phenotypes and high organ transplantation sensitivity; as a result, the prevalence of severe hyper acute rejection by preformed DSAs has decreased significantly (7). Accordingly, Ng et al. summarized desensitization protocols and complications using rituximab, bortezomib, eculizumab, and alemtuzumab, and reported promising graft survival in patients across various institutes (78). However, complications included anemia and thrombocytopenia, likely reflecting myelosuppression by these agents. In addition, various infections in some cases were detected, including cytomegalovirus (CMV), BK virus, and Epstein–Barr virus (EBV), indicating that desensitization therapy disposes patients to an increased risk of opportunistic viral infections. In addition, it was reported that induction with T-cell depleting agents (anti-thymocyte globulin) was closely associated with CMV, EBV, and BK polyomavirus (BKV) infections in comparison with IL-2a receptor antagonists (anti-CD25) (79). Therefore, the use of T-cell depleting agents should be avoided as an immunosuppressive reagent or induction. If possible, the use of IL-2a receptor antagonists or no induction should be considered (79, 80). Additionally, it was expected that these virus infection may contribute to the activation of immune responses in transplanted organs, and dose reductions of immunosuppressive agents may activate immune reactions to graft antigens.

To address this issue, prediction and early detection of viral infections is critical, and could be used to inform doses reductions of immunosuppressive agents. Concomitant administration of preventive and therapeutic antiviral agents is also critical in the management of these patients.

#### Desensitization Therapy

Prior to the introduction of rituximab, plasmapheresis and splenectomy were long recommended as desensitization therapies for patients with ABO incompatible kidney transplants. Subsequently, rituximab was shown to inhibit the onset of ABMR without splenectomy. Rituximab is a monoclonal antibody (mAb) against the protein CD20, which is expressed in immature and mature B cells. However, because CD20 is not expressed on PCs, rituximab may not inhibit the production of DSAs by PCs. In addition, recent studies show varying effects of rituximab on B cell phenotypes, with higher sensitivity of naïve B cells than memory B cells (81). Thus, although rituximab suppresses immune activation and may not provide protection from infection, memory B cells may remain viable.

In addition, posterior reversible encephalopathy syndrome (PRES) and acute respiratory distress syndrome (ARDS) was reportedly increased in patients treated with rituximab as severe adversity effect (82). These data warrant further clarification of the depletion mechanisms of rituximab in B cells.

Bortezomib is a proteasome inhibitor that was developed as a treatment for multiple myeloma, and the effectiveness of this agent against transplant rejection was reported in 2008. These studies showed downregulated immune responses to donor antigens, recovery of graft function, and long-term suppression of serum antibody levels. However, inhibition of the proteasome by bortezomib may be detrimental to healthy cells (83–88).

As an alternative, eculizumab is a recombinant humanized monoclonal IgG2/4 antibody that suppresses complement activation and inhibits production of C5, which is the final product of the complement pathway and activates inflammatory responses and ultimately results in apoptosis of infected cells (89). Accordingly, treatments with this agent led to severe infectious diseases, including meningitis (90).

Finally, alemtuzumab is a recombinant DNA-derived humanized IgG1 kappa mAb that is directed toward CD52 and is used is used to treat B-cell chronic lymphocytic leukemia (B-CLL) and multiple sclerosis patients, warranting consideration for the treatment of ABMR. As adverse effect, it has been associated with infusion-related events (91, 92).

#### Infection as a Trigger of Rejection Cytomegalovirus Infection as a Trigger of Rejection

Cytomegalovirus is among the most common infections after solid-organ transplantation, and results in significant morbidity, graft loss, and adversity. Although numbers of CMV-seronegative (R−) cases have increased recently in healthy subjects, those with organ transplants from CMV-seropositive donors (D+) are at the highest risk of primary CMV disease, which can easily become serious causing the reactivation of latent virus transmitted in the allograft (93, 94). Additionally, a close relationship between CMV infection and allograft rejection has been reported in CMV D+/R− liver and kidney transplant patients (93, 95).

#### *Laboratory Diagnosis of CMV*

*Nucleic Acid Testing.* Nucleic acid testing (NAT) is widely used to detect and quantify CMV RNA and DNA*.*

*Serology.* Serological analyzes allow risk stratification of patients during the pre-transplant screening phase on the basis of tests for CMV IgG antibodies in both donors and recipients, and can indicate the presence of latent infection*.*

*Antigenemia.* The antigenemia assay detects the CMV pp65 antigen in infected leukocytes from peripheral blood, and has been used for rapid diagnosis of CMV infections in transplant recipients (96)*.*

#### *Treatment of CMV*

In a previous study, valganciclovir was found to be more effective than oral ganciclovir at preventing CMV disease in solid organ transplant recipients (97), suggesting that extension of valganciclovir prophylaxis to 200 days may benefit high risk (D+/ R−) kidney recipients. Following transplantation, CMV disease is predominantly treated using intravenous (IV) ganciclovir (5 mg/ kg every 12 h) and oral valganciclovir (900 mg twice daily) (98).

#### BK Polyomavirus Infection as a Trigger of Rejection

More than 90% of healthy subjects become infected with BKV (99, 100), which is the major cause of polyomavirus-associated nephropathy (Py-VAN) and presents a 1–15% risk of allograft failure in kidney transplant patients (101–106). And it has been reported that BKV-activated antibody reactivity in recipients at the onset of immunosuppression (107). However, although number of BKV-seronegative (R−) cases has increased recently in healthy subjects, these patients are the most susceptible to BKV disease following transplantation from BKV seropositive donors (D+) (108, 109).

#### *Laboratory Diagnosis*

Screening for BKV replication should be performed at least every 3 months during the first 2 years after transplantation, and then annually until the fifth year.

*Nucleic Acid Testing.* Nucleic acid testing in polymerase chain reaction (PCR) is used to detect amplifications of BK DNA*.*

*Urine Cytology.* Urine cytology is sufficient to detect decoy cells, which are associated with BKV induced organ failure*.*

#### *Treatment of BKV*

First, reduction of immunosuppression should be considered (110, 111). In patients with sustained high-level plasma BKV loads despite dose reductions of immunosuppression agents, administration of antiviral agents (*Cidofovir)*, and a replacement for mycophenolic acid (*Leflunomide)*, intravenous immunoglobulin (IVIG), and anti-mycotic agents (*Fluoroquinolones*) should be considered.

#### Epstein–Barr Virus Infection as a Trigger of Rejection

Epstein–Barr virus contributes to the pathogenesis of posttransplant lymphoproliferative disease (PTLD) occurring cases early after transplantation in more than 90% of the cases, and small intestine transplantations are associated with higher risks than heart, lung, and liver transplantations (112, 113). The close relationship between EBV infection and ABMR has been reported in heart transplantation (114).

#### *Laboratory Diagnosis*

*Nucleic Acid Testing.* Epstein–Barr virus DNA monitoring for EBV D+/R− recipients should be recommended, with continued EBV load screening every 3–6 months until 2–3 years after transplantation. This monitoring is particularly important for EBV-seropositive recipients with intestinal transplants, and monitoring of EBV DNA every 2–4 weeks in the first 3 months should be performed, monthly until 6 months post-transplantation, and then every 3 months until the end of the first year*.*

#### *Treatment of EBV*

Antiviral prophylaxis for high risk patients (EBV D+/R−) is considered in some centers (99).

Treatment with acyclovir, ganciclovir, and IVIGs has shown some benefits in the prevention of PTLD among EBV-seronegative recipients who their donors are EBV-seropositive (113).

## THE ROLE OF *DE NOVO* DSA IN THE PATHOGENICITY OF GRAFT INJURY

Recent reports show that DSAs play an important role in ABMR onset, and this has been shown by highly sensitive monitoring of HLA antibodies in the sera (11–13). However, DSAs may be absorbed into transplanted organs during the early phases of antibody production (115) (**Figure 2**). Accordingly, in the Guidelines of the Transplantation Society (TTS), post-kidney transplant DSA monitoring is not recommended for all patients beyond the first year (116). Hence, to avoid the influence of absorption, antibody production from PCs has been analyzed *in vitro*, because these antibodies may not be influenced by the absorption and provide us with further detailed illustrations that are available to clarify how these antibodies are produced in organ recipients. However, PCs are seldom found in blood and predominate in bone marrow and secondary lymphoid tissues, techniques for differentiating B cells into PCs, are required to investigate antibody production (117–119).

phases of antibody production.

## *In Vitro* B Cell Assays

Determination of HLA antibodies in supernatants of cultured B cells can better inform ABMR management than those in sera. Therefore, some researchers have suggested that if peripheral B cells could be differentiated into PCs *in vitro*, then the *in vitro* differentiation of peripheral B cells into PCs may facilitate the control of ABMR. However, unlike *in vitro* T cell assays that have long been used to control T cell-mediated rejection (TCMR), primary cultures of B cells are difficult to maintain and *in vitro* B cells assays have not long been established. Although memory B cells were reportedly differentiated into APC *in vitro* (120), these short comings require further improvements in the ease and convenience of B cell culture, and subsequent assay development that can be used to detect HLA antibodies in B cell culture supernatants to ultimately prevent transplant rejection.

Moreover, there are important points to detect HLA antibodies from the B cell culture supernatant. Peripheral B cells include naïve B cells and memory B cells (121, 122); and these B cells derived from PCs survive for varying durations and produce antibodies. However, HLA antibodies that cause ABMR are mainly produced by memory B cells (123–125), warranting establishment of *in vitro* B cell assays in which memory B cells are selectively differentiated into PCs *in vitro* and are used to produce antibodies. In addition, many reports have showed that long-lived PCs, which produce antibodies in the bone marrow for long periods, play an important role in ABMR (126–129). Therefore, to monitor the progression of antibody-mediated diseases, *in vitro* culture systems, in which B cells are differentiated to their terminal stage (long-lived PCs), are urgently required. On using a clinical specimen, the volume of B cells in peripheral blood is very low following immunosuppression or particularly when we could collect B cells from recipients who experienced desensitization therapy.

However, feeder cells can strongly activate human B cells to proliferate and differentiate in a cell–cell contact-dependent manner in these cases. Thus, *in vitro* B cell assays of HLA antibodies from the cultured supernatants may lead to drug sensitivity tests that are similar to those for T cells, may contribute to clinical applications of personalized immunosuppression, and the development of new immunosuppressant agents that control ABMR.

# *In Vitro* Memory B Cell Assays

We found a report about immunosuppressive agent susceptibility for the differentiation of human B (CD19+) cells *in vitro* with a combination of IL-21, phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN), histidine-tagged soluble recombinant human CD40 L and anti-polyhistidine mAb (130). IL-21 is produced by follicular helper T cells (131), which synergistically induce maximum Blimp-1 upregulation and optimal PC differentiation with CD40 L (132). TLR9 agonist CpG-ODN activates B cells proliferation and promotes PCs differentiation (133). This culture system induced IgG production but could not sustain the survival of PCs for a long period. It might indicate that the other cytokines play an important role in human B cells differentiation into mature PCs *in vitro*, because other groups have reported that APRIL and the B cell activating factor (BAFF) would support the survival of PBs and PCs recently (134–137). In addition, a previous report has shown that CD27+ memory B cells could be differentiated into long-lived PCs with supernatants from bone marrow stromal cell line M2-10B4 (138), which support the longterm culture of human bone marrow stem cells. The mechanisms by which M2-10B4 cells contribute to PCs survival has yet to be revealed, but it is suggested that CD27+ memory B cells demand well-balanced support from stromal cells (139–142). In addition, different environments or signal transmission might be required for the differentiation of CD27− naïve and CD27+ memory B cells into mature PCs. Therefore, we should improve the *in vitro* B cell assay to sustain CD27+ memory B cell-derived PC survival for a long-term selectively. For example, we should examine how any humoral factors, including growth factor or any cytokines from activated T cells could affect CD27+ memory B cell growth and survival *in vitro*, while referring to the reports that helper T-cells may mediate CD27+ memory B cell differentiation into PCs *in vivo* (143).

## Risk Factors of ABMR from *De Novo* DSA

Not all DSAs participate in ABMR and transplanted organ prognosis (7), and although C1q binding DSAs are reported risk factors for ABMR onset, further studies of DSA characteristics are required to identify those with prognostic value. In addition, various other factors influence transplanted organ prognoses (ABMR onset, graft survival) and require further investigation. About the risk factors for graft loss, thrombotic microangiopathy (TMA), glomerulopathy, C4d deposition, and chronic injury change in histopathological diagnoses were reported.

As other factors besides histopathological findings, a history of subclinical ABMR and TCMR and a decline of graft function could be risk factors. This might indicate that a graft would fail with high probability when the humoral immune response toward a donor-specific antigen has proceeded to an irreversible stage.

#### C1q Binding DSA

C1q appear in the beginning of the classical complement pathway, and C1q binds directly to antigens and initiates classical complement pathway activation. Subsequent C1q-activated reactions include (i) antigen binding, (ii) binding to C-reactive protein, and (iii) binding to antigen–antibody complexes, and can lead to the activation of C3 convertase and the degradation of C3 to C3b and C3a (144, 145). Of these, C3b is the main effector of the complement pathway, while C3a activates inflammatory responses. Indeed, C1q may play important roles in the activation of inflammatory reactions against grafts. Accordingly, C1q binding to DSAs reportedly influences the frequency of ABMR onset and glomerulopathy in solid organ transplants, leading to increased chances of graft failure. Thus, binding of C1q to DSAs may be highly predictive of graft prognosis, warranting the development of interventions that decrease the presence of C1q binding DSAs. The C1qScreen™ (One Lamda) is a reliable tool for distinguishing complement-binding antibodies from non-complement-binding ones, and is widely applied using Luminex-based LABScan™ 100 flow fluorescence analyzers to determine relative amounts of C1q binding antibodies. The C1qScreen™ in combination with the Luminex-based LABScan™ can indicate the relative amount of C1q bound to DSAs and provides us with useful information from the sera (146). In addition, in C1q-positive cases despite being DSA-negative, graft survival is poor. It suggested that C1q could affect the transplanted organ prognosis by itself.

#### DSA Characteristics