Characterization of the C1q-Binding Ability and the IgG1-4 Subclass Profile of Preformed Anti-HLA Antibodies by Solid-Phase Assays

Abstract

Humoral alloimmunity, particularly that triggered by preformed antibodies against human leukocyte antigens (HLA), is associated with an increased prevalence of rejection and reduced transplant survival. The high sensitivity of solid phase assays, based on microbeads coated with single antigens (SAB), consolidated them as the gold-standard method to characterize anti-HLA antibodies, ensuring a successful allograft allocation. Mean fluorescence intensity (MFI) provided by SAB is regularly used to stratify the immunological risk, assuming it as a reliable estimation of the antibody-level, but it is often limited by artifacts. Beyond MFI, other properties, such as the complement-binding ability or the IgG1-4 subclass profile have been examined to more accurately define the clinical relevance of antibodies and clarify their functional properties. However, there are still unresolved issues. Neat serum-samples from 20 highly-sensitized patients were analyzed by SAB-panIgG, SAB-IgG1-4 subclass and SAB-C1q assays. All 1:16 diluted serum-samples were additionally analyzed by SAB-panIgG and SAB-IgG1-4 subclass assays. A total of 1,285 anti-HLA antibodies were identified as positive, 473 (36.8%) of which were C1q-binding. As expected, serum-dilution enhanced the correlation between the C1q-binding ability and the antibody-strength, measured as the MFI (r neat = 0.248 vs. r diluted = 0.817). SAB-subclass assay revealed at least one IgG1-4 subclass in 1,012 (78.8%) positive antibody-specificities. Among them, strong complement-binding subclasses, mainly IgG1, were particularly frequent (98.9%) and no differences were found between C1q- and non-C1q-binding antibodies regarding their presence (99.4 vs. 98.5%; p = 0.193). In contrast, weak or non-C1q-binding subclasses (IgG2/IgG4) were more commonly detected in C1q-binding antibodies (78.9 vs. 38.6%; p < 0.001). Interestingly, a strong association was found between the C1q-binding ability and the IgG1 strength (r IgG1dil = 0.796). Though lower, the correlation between the IgG2 strength and the C1q-binding ability was also strong (r IgG2dil = 0.758), being both subclasses closely related (r IgG1−IgG2 = 0.817). We did not find any correlation with the C1q-binding ability considering the remaining subclasses. In conclusion, we demonstrate that a particular profile of IgG subclasses (IgG1/IgG3) itself does not determine at all the ability to bind complement of anti-HLA antibodies assessed by SAB-C1q assay. It is the IgG subclass strength, mainly of IgG1, which usually appears in combination with IgG2, that best correlates with it.

La historia de un trasplante renal previo es el evento más importante para identificar subclases de IgG de los anticuerpos anti-HLA y positividad del ensayo C3d

“…Our findings resembles those recently published by Navas et al demonstrating a close relationship between the circulating antibody strength, the presence of a mixture of IgG subclasses, beyond the quasy omnipresent IgG1, and the complement-binding ability measured by C1q. 32 Important to mention are the results obtained in this study in relation to the low frequency detected for both IgG subclass identification and C3d positivity which corresponded to 20.7% and 12.2%, respectively, of the total 1,771 HLA-Abs detected by the three methods used. Whether methodological factors could took place, at least in part in these results, cannot be ruled out despite the methods followed were exactly as the manufacturer indicate.…”

Plasma cell biology: Foundations for targeted therapeutic development in transplantation

“…Optimal C1q engagement by the complement‐binding region of antibodies is mediated when anti‐HLA antibodies are bound to cell surface HLA molecules (particularly if HLA molecules are highly expressed) and arranged in a hexagonal pattern. Once bound to cell surface molecules on the endothelium, antibody subclasses IgG1 and IgG3 in particular, bind the complement component C1q, and initiate the classical complement cascade 82,83 . This results in the recruitment of leukocytes which further induce inflammation and allograft damage as well as direct lysis of the targeted cell through complement‐dependent cytotoxicity.…”

Approaches for the characterization of clinically relevant pre‐transplant human leucocyte antigen (HLA) antibodies in solid organ transplant patients

“…Additionally, there is a maximum detection limit ~20,000 MFI using the currently ubiquitous Luminex ® 100/200™, although the newer Luminex ® FLEXMAP 3D platform has been reported to have a greater detection range (up to 400,000 MFI), which may reduce saturation errors in the future (Gebel & Bray, 2019). The relative impact of saturation can only be proven by retesting following dilution of sera (McCaughan et al, 2019;Navas et al, 2019;Tambur & Wiebe, 2018). Similarly, the effects of prozone can be reduced by dilution or pre-treatment of sera using heat inactivation, ethylenediaminetetraacetic acid (EDTA) or dithiothreitol (DTT) to remove blocking factors (i.e., complement and IgM antibody respectively) (Anani et al, 2016;Battle et al, 2017;Courant et al, 2018;Gebel & Bray, 2019;Guidicelli et al, 2013;Middleton et al, 2014).…”

“…The association between a high MFI value and complementfixing has been reported as a confounding factor for both the C1q and C3d assays (Cioni et al, 2017;Claisse et al, 2017;Courant et al, 2018;Gautier Vargas et al, 2019;Kamburova et al, 2018;Karahan et al, 2017;Ko et al, 2018;Moreno Gonzales et al, 2017;Schaub et al, 2014;Tait et al, 2013;Yell et al, 2015), although antibodies with high MFI value are not always reported to be complement fixing (Chen et al, 2011;Navas et al, 2019;Zeevi et al, 2012). As the density and avidity of bound antibody affects the efficiency of C1qbinding, it would suggest that lower titre antibodies (reported as 1:34 to 1:64 titre) (Tambur & Wiebe, 2018) may give a false negative result for assessment of complement-fixing ability (Filippone & Farber, 2016;Schaub et al, 2014;Tambur et al, 2015;Thurman et al, 2019).…”