Investigating the relationship between class I HLA-specific immunoglobulin-G subclasses, Pan-IgG single antigen bead assays and complement mediated interference in sera from renal transplant recipients

Highlights

• We performed a detailed analysis of IgG subclass and pan-IgG anti-HLA antibodies in a post-transplant population, using single antigen bead technology

• We determined a 1 in 10 dilution to be the optimum pan-IgG comparator assay for a subclass analysis

• A summed subclass mean fluorescence intensity identified potential subclass failures

• MFI cut-offs for IgG subclasses should be used with caution given many inhibitory influences

Abstract

Introduction

Antibody mediated rejection is the leading cause of kidney transplant failure. Not all antibodies are harmful and some may be protective. Immunoglulin Gs, of which there are four subtypes, are detected by single antigen bead testing. The aims of this study were to characterise the IgG subclass profiles for class I HLA-specific antibodies in an uncensored post-transplant population and to determine the underlying relationship between reactivity patterns and MFI cut-offs with the pan-IgG assay.

Methods

Patients were recruited to the study who were transplanted in our centre between 2009 and 2014. Prospectively stored post-transplant serum initially underwent a Labscreen Mixed assay and those positive for class I HLA-specific antibody underwent standard SAB testing, EDTA, 1 in 10 dilution and IgG subclass modifications using the Luminex platform. A total of 4947 bead reactions from 51 patients were analysed.

Results

A 1 in 10 dilution was used as a comparator pan-IgG assay for summed subclass and individual subclass linear regression analyses. Using a dilution to standard assay ratio we characterised all reactions for prozone potential i.e. how likely there is to be inhibition related to complement complex formation. We stratified samples into degrees of association and were able to determine suggested MFI thresholds of Log 5.35 for the dilution assay and Log 5.05 for the summed subclass assay when considering a Log MFI of 6.9 (1000) in the standard assay. Using individual subclass dominant reactions (>70%) we were able to determine linear relationships between the 1 in 10 dilution pan-IgG assay and the individual subclass assays (excluding prozone potential reactions for IgG1/3) enabling us to suggest Log MFI thresholds of 5.03, 3.58, 4.3 and 4.05 respectively for IgG1–4.

Discussion

We recommend a 1 in 10 dilution as the optimum pan-IgG comparator assay for a subclass analysis. We advocate the utilisation of the summed subclass assay to determine overall relationships and potential subclass failures. Following others, we recommend serum pre-treatment of the subclass assays to mitigate prozone. We suggest cut-offs for each IgG subclass which should be used with caution given the many inhibitory influences which may include competitive inhibition for bead binding, IgM and IgA interference and under-representation of specific subclasses on the bead panel.

Introduction

Rejection of organs due to HLA-specific antibody mediated rejection (AbMR) is the leading loss of kidney transplants worldwide [1]. Reported rates of donor specific antibody (DSA) post-transplant suggest a 10–12 year incidence of 25–27% [2,3].

AbMR has both clinical and subclinical phenotypes [4], the former being typified by an acute, rapid deterioration in graft function and the latter having a more indolent progression over years. Both manifestations have a deleterious effect on graft function and survival and, unlike cellular rejection, lack efficacious treatments [1].

Not all transplant recipients with DSA will go on to develop graft dysfunction: one study in kidney transplant recipients suggested a 10 year graft survival of 59% in patients with DSA compared to 96% in patients without [5], with another study describing eight year graft survival of 60.6% and 78.4% for patients with strong or weak preformed DSA compared to 82.5% without [6], indicating that not all DSA have pathological consequences [7]. Thus, a better understanding of the characteristics of antibodies implicated in AbMR is necessary to elucidate the pathological processes leading to graft injury and loss.

In response to antigenic stimulation, the first immunoglobulin (Ig) synthesised is IgM, then IgD followed by IgG3, which has a half-life of 7 days [7,8]. Assuming appropriate co-stimulation and T-cell help (from TH² helper T-cells), class switching then occurs sequentially from IgG3 → IgG1 → IgA1 → IgG2 → IgG4 → IgE → IgA2. IgG3 and IgG1 are potent activators of complement, IgG2 can only do so at high antibody concentration and IgG4 cannot activate it [7].

Single antigen bead (SAB) assays target IgGs, and have been used to enhance pre-transplant cross-matching and post-transplant investigation of rejection. The standard SAB assays identify all subclasses of IgG (pan-IgG), of which there are 4 (IgG1–4) directed against HLA. However, there are inhibitory influences, the best characterised of which is the so called “prozone effect” which attenuates the MFI in the standard assay. Complement mediated interference (CMI) is an important cause of prozone and is driven by a high concentration of HLA-specific IgG antibody forming complement-mediated complexes which inhibit binding of the IgG reporter antibody. A number of assay modifications can abrogate its effect [9].

There is a small body of literature about the prevalence and role of IgG subclasses following solid organ transplantation. The studies are heterogeneous in subject selection, design and outcome measurements. Generally IgG1 predominates post-transplant [4,[10], [11], [12], [13], [14], [15], [16], [17]]. The pathological effects of the subclasses post-transplant are less well described. IgG4 is implicated in subclinical AbMR [4], graft loss [18] and acute rejection [18] in kidney transplants and poorer histological findings in paediatric liver transplant biopsies [10]. IgG3 is associated with graft loss in liver [11] and kidney transplantation [3,4,14,18,19] as well as IgG1 in liver transplantation [20].

The range of MFI thresholds (i.e. nominal cut-offs beneath which clinical relevance is unlikely and are thought of as negative reactions) in these subclass studies also varies, but they are consistently lower than thresholds typically adopted for the standard assay [9]. Several studies reported a subclass MFI threshold of 500 [4,[10], [11], [12],15,21,22] whereas others reported much lower MFIs of <150 [13,17,18,23]. Reasons for the MFI attenuation with the subclass secondary antibody are unclear. Only one study examines the summed MFIs of all four subclasses [10] without exploring how the individual subclass, summed subclass and pan-IgG MFIs relate. None of these studies attempt to profile CMI in, nor the proportional representation of, the subclass assays. Neither more do they address the relationships between the pan-IgG assay and the subclass assays which we believe offer an important area of investigation.