Discussion
This study documents the IgG subclass cross-reactivity pattern of several IgG subclass-specific mAbs that are used for the detection of DSA subclasses in prospective and actual transplant recipients. This study is the first to document an extensive titrimetric analysis of IgG subclass cross-reactivity pattern that is dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets. While the results of this study have a minimal impact on the general clinical findings about IgG subclass-specific DSAs, the awareness of such cross-reactivity may help the refinement and understanding of the complex distribution of IgG subclass-specific DSAs found in transplant patients (10,15,19,20). Surprisingly, only the anti-human-IgG3 mAb (clone: HP6050) was truly mono-specific at all concentrations tested, where it recognized only IgG3 proteins and not IgG1, IgG2 or IgG4 proteins. All other mAbs tested were binding to IgG subclass proteins in addition to their respective immunogen, thereby being cross-reactive. This extensive cross-reactivity pattern of the different IgG subclass-specific mAbs was not realized before, although minimal cross-reactivity was detected for anti-IgG1 and -IgG2 mAbs particularly (15,17). Only a few studies have validated the modified single antigen beads (SAB) assay procedure for the screening of IgG subclass DSA (14–17,21). The validation of the modified SAB assay relies on several parameters, such as custom IgG subclass-specific coated-beads, IgG subclass-specific mAbs concentrations, that vary across HLA laboratories. Nonetheless, this study supports the use of Luminex® multiplex platform as an effective approach to assess IgG subclass cross-reactivity of currently available and newly generated mAbs.
In allograft recipients, several studies have demonstrated a clinical impact of IgG subclass DSAs on allograft survival, outcomes, and adverse events, where IgG3 DSAs have been shown to have the most deleterious impact. Indeed, the presence of IgG3 DSAs was associated with acute ABMR, shorter time to rejection, allograft failure (10), poor graft survival (21), and biopsies positive for C4d (17) in adult kidney recipients, and with a higher risk of graft dysfunction in pediatric kidney recipients (22). In adult liver recipients, IgG3 DSAs were associated with an increased risk of graft loss (16) and with death (23). These findings are not impacted by the use of the anti-human-IgG3 mAb (clone: HP6050) since it is mono-specific to IgG3 proteins, and thus considerably reduces the rate of false-positive IgG3 recipients. However, IgG3 DSA-positive recipients will most likely be IgG1 DSA-false-positive when cross-reactive anti-human-IgG1 mAbs (clone: HP6001 and 4E3) are used at high concentration or when serum concentration of IgG3 DSA is high.
Regarding IgG4 DSAs, their presence pre-transplant, in kidney recipients, was an independent predictor of early rejection, graft failure, and graft survival (21), while their presence post-transplant was associated with subclinical ABMR (10). In pediatric liver recipients, IgG4 DSAs are associated with a histopathological and transcriptional features indicative of active controlled alloimmune response (T cell mediated rejection) (14). The IgG4 DSA false-positive rate in these findings should be low since the anti-human-IgG4 mAb (clone: HP6025) cross-reacts with IgG2 proteins at high concentrations only, and possibly because IgG2 do not constitute a large proportion of IgG DSAs (8). In contrast, IgG1 and IgG2 false-positive rates may be more impacted by our findings, where IgG1 and IgG2 results should be viewed in the context of cross-reactivity, as previously suggested (17). Although recipients with IgG1 DSAs only can clearly be identified by the anti-IgG subclass mAbs pattern of reactivity described in this study, and IgG1 DSAs unequivocally represents the predominant HLA subclass (10,14–17,19–22,24), recipients with IgG2 and/or IgG3 DSAs are likely to exhibit IgG1 DSA false positivity as a result of anti-IgG1 mAbs cross-reactivity (Table 2). Similarly, anti-IgG2 mAbs exhibit the greatest cross-reactivity, particularly clone 31-7-4 (14), and patients with IgG1, IgG3, and/or IgG4 DSAs are likely to be IgG2 DSA-false-positive. In support, IgG2 DSA-positive only recipients are absent (10,17,21,24) or extremely rare (11,15,20,22). Therefore, the distribution of IgG subclass DSAs in transplant recipients should be reevaluated in light of our findings, in order to confirm, for example, whether the presence of specific IgG subclass DSAs are associated with the type of immunization (transplantation, transfusion, pregnancy) in HLA-sensitized patients (15,19), or whether the presence of multiple IgG subclass was associated with chronic rejection in adult liver recipients (16). Overall, our study confirms that IgG subclass reagents, particularly IgG subclass-specific mAbs, must be optimized before the implementation of IgG subclass DSA analyses in clinical practice (10).
Differences in the generation of IgG subclass-specific beads, anti-IgG subclass mAb concentration, and anti-IgG subclass mAb clone selection could explain the differences in MFI reported in different cross-reactivity assessments (14–17,21). Nonetheless, several approaches may contribute to the validation and standardization of IgG subclass DSA screening in anticipation of newly developed anti-IgG subclass mono-specific mAbs. First, our study provides the detailed description for the generation of beads covalently coated with specific IgG subclass proteins. This process facilitates the control of the amount of IgG subclass protein coated onto the beads, which is an important factor in the observed cross-reactivity of the different anti-IgG subclass mAbs in our study. Covalent and non-covalent IgG-protein coating are two methods commonly used to generate IgG subclass-specific coated-beads but parameters, such as the amount of IgG subclass protein coated onto the beads, are usually not reported. Secondly, there is no consensus for the working concentration of each IgG subclass mAbs with maximum of an 80-fold difference between reports (Table 1); however, our study demonstrates that the concentration of the IgG subclass mAbs is a critical factor that determines the relationship between maximum MFI detection capacity (sensitivity) and IgG subclass protein cross-reactivity (specificity). We demonstrated, for cross-reactive IgG subclass mAbs, that a high concentration increases the sensitivity while reducing the specificity, conversely a low concentration reduces the sensitivity while increasing the specificity. Moreover, our results confirm that the affinity and avidity varies between anti-IgG subclass mAbs, as the direct MFI comparison of each mAb at different concentration does not reflect relative amount of IgG subclass protein, and thus supports the use of a unique working concentration for each anti-IgG subclass mAbs. Thirdly, our study suggests that the use of clones HP6001, HP6014, HP6050 and HP6025 to detect IgG1, IgG2, IgG3, and IgG4 DSAs, respectively, is the optimal anti-IgG subclass mAbs combination that maximizes the sensitivity and specificity of subclass DSA screenings using Luminex® SAB assays. In terms of cross-reactivity, only the anti-IgG4 mAbs, HP6025 and HP6023, did not exhibit notable differences; however, HP6025 seems most suitable to use with HP6001, HP6014, and HP6050. The affinity of HP6025 for its target is closer to that of other IgG subclass mAbs, whereas HP6023 appears to have a higher affinity since the concentration of HP6023 was lowest to reach saturation of the beads compared to other IgG subclass mAbs; moreover, HP6025 was used more frequently to detect IgG4 DSAs, thus providing consistency for the comparison of MFIs with the reports of previous studies. Last, the disparity in the methods defining the threshold for IgG subclass DSA positivity warrants further investigations. Yet, the appropriate threshold for positivity should be determined for each IgG subclass mAb separately. In spite of the well-known limitations of the SAB assays (25,26), variations in the modified protocols for the IgG subclass DSA screening impair the direct comparison of different studies’ results, particularly for MFI strengths.
The Luminex® SAB assay has not been approved as a quantitative assay by the US Food and Drug Administration to measure the concentration of DSAs (27). However, it is evident that allo-sensitization, defined by a marked increase of IgG DSA production, is detected by the SAB assay. Extensive serum titration analyses revealed that some DSA still show high strength (MFI>15,000) at a serum dilution of 1:1024 (28). Similarly, at a serum dilution of 1:16, Navas et al. demonstrated that the IgG subclass DSA strength correlated with the ability to bind complement particularly when IgG1 DSAs were accompanied with IgG2 DSAs (20). In addition, C1q-binding DSAs usually exhibit high titers in the regular SAB assay (28), and anti-IgG2 mAb (clone: 31-7-4, at 20µg/mL) cross-reacted with IgG1 proteins at high concentrations (1µg and 2µg per 1x106beads); therefore, it suggests that serum DSA concentrations (or density of bead-bound DSAs) clearly impact the cross-reactivity level of IgG subclass mAbs and prevent the clear distinction of patients’ IgG subclass DSA distribution. Furthermore, weak DSAs (MFI<2,000) detected with the regular SAB assay, can be negative in the modified IgG subclass specific SAB assay (8), particularly if the concentration of the anti-IgG subclass mAb is low. Similarly, weak HLA antibodies (MFI<5,000) detected with the conventional PE-conjugated polyclonal anti-human-IgG had a significantly lower MFI when detected with a PE-conjugated anti-human-IgG mAb, while the opposite was true for de novo DSA particularly (18). In spite of the extreme variability of serum IgG subclass DSA concentrations and the impact on DSA detection, the goal in validating the use of PE-conjugated mAbs in IgG subclass DSA screening is directly related to the serum dilutions used for testing, where optimal conditions increases the sensitivity and decreases the cross-reactivity of the assay. The refinement of patients’ IgG subclass distribution assessment can benefit from serum dilution experiments, despite the cost-prohibitive nature of serum dilution screening using the SAB assay.
This empirical analysis of IgG subclass mAbs cross-reactivity has a few limitations. First, interfering factors present in the serum may affect the cross-reactivity of IgG subclass mAbs. A recent study purified serum IgG to remove factors that can interfere with the SAB assay and thus may enhance MFI signal of DSA (14); however, the purification of serum IgG reveals an extensive HLA reactivity that was masked in the native sera (29), which may affect IgG subclass DSA distribution of purified serum IgG samples and further studies are needed to address this problem. Second, besides the <90% homology between IgG subclasses, which makes the generation of mAbs delicate for producing mono-specific ones, the presence of different IgG subclass allotypes further complicates this problem. Since different IgG subclass allotypes may be implicated in their immunogenicity by inducing allotype-specific antibodies (30), it is important to verify that each IgG subclass-specific mAbs recognize each allotypes equally, and that there is no impact on IgG subclass cross-reactivity. Last, we have tested only one lot for each IgG subclass mAbs, but Cicciarelli et al. have suggested that different lots of mAbs may have different pattern of cross-reactivity (17). Indeed, the anti-IgG1 (clone: HP6001) was shown to be cross-reactive to IgG4, and, interestingly, we observed cross-reactivity to IgG2 and IgG3, but not to IgG4, for the same clone from a different source.
In conclusion, we have characterized and validated the IgG1-4 subclass cross-reactivity pattern of 8 anti-IgG subclass-specific mAbs. To our surprise, only one anti-IgG3 specific mAb (clone: HP6050) was mono-specific, whereas all other anti-IgG subclass mAbs were cross-reactive. Importantly, IgG subclass mAb cross-reactivity is dependent on both their concentration and the concentration of the IgG1-4 subclass protein targets. Despite the observed cross-reactivity of certain mAbs, unique patterns of reactivity, which correspond to a unique IgG subclass composition, can be identified. Although the impact of IgG subclass DSAs on allograft outcomes that was previously demonstrated is not critically influenced by our data, the dilution of serum samples and IgG subclass-specific mAbs must be further optimized before their implementation of IgG subclass DSA screening in allograft recipients.