Effects of weak/non‐complement‐binding HLA antibodies on C1q‐binding

Abstract

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory.

Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: A systematic review and meta-analysis

“…In contrast to MFI that was reported in most of the studies in this meta-analysis, anti-HLA DSA level determined by titer of antibody correlated with complement-fixing ability [ 22 , 74 ]. In addition to the requirement of minimum titer of DSAs (>1:16) to be complement fixing, the composition of IgG subtypes may also influence the complement-binding capacity [ 48 , 75 ]. Therefore, C1q, C3d and IgG3 assays provide additional insights beyond the DSA strength/titer.…”

Caveats of HLA antibody detection by solid‐phase assays

“…Independent of their clinical relevance, these antibodies—not monitored by the standard anti‐IgG pan —may have an influence on MFI, as they are competing for binding on SAB and thus may decrease IgG‐derived MFI . Moreover, the final level of MFI is influenced by concentration, affinity, and incubation time of the used detection antibody conjugate .…”

“…Notably, about 98% of DSA contain either IgG1 or IgG3 subclasses and hence have the intrinsic capability to bind complement . The major determinant of C1q or C3d binding on SAB is the amount of DSA, which can be approximated by the MFI value . It is not surprising that most studies found a very strong correlation of the MFI value and C1q or C3d binding .…”

“…High amount of IgG2 and/or IgG4 can block the effect of IgG1 and/or IgG3 subclasses if the antibodies target the same epitope. By contrast, these subclasses can act synergistically if they target different epitopes on the same HLA molecule . It has also been shown that an expansion of DSA to IgG2/IgG4 subclasses rather indicates an advanced immune response .…”

Technical challenges and clinical relevance of single antigen bead C1q/C3d testing and IgG subclass analysis of human leukocyte antigen antibodies

“…Hönger et al . used IgG1–4 subclass variants of a chimeric mouse/human monoclonal HLA class II antibody to show that IgG2/IgG4 antibodies profoundly inhibited C1q binding when present at onefold to fourfold higher levels than IgG1 and IgG3, and completely hampered C1q binding when present at a level of 10‐fold excess.…”