Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of protease- and IgG subclass-restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus pyogenes (IdeS)- or pepsin-generated F(ab') fragments of all four human IgG subclasses was determined. AHA reactivity against one or more out of eight F(ab') targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and 81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create the F(ab') targets, as confirmed by inhibition experiments with F(ab') fragments and hinge peptides. Reactivity against IdeS-generated F(ab') targets was found most frequently, whereas reactivity against pepsin-generated F(ab') targets better discriminated between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab')s was detected. The most discriminatory AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ≥5.8-fold higher than in HDs/SLE, and significantly higher levels (p < 0.0001). Cross-reactivity for F(ab')s generated from different IgG subclasses was only observed for subclasses having homologous F(ab') C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass- and protease-restricted hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory or infectious conditions and may be markers of, and participants in, such processes.

T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201–binding WT1126–134 peptide were generated from both HLA-A*02–positive (self-HLA–restricted) and HLA-A*02–negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA–restricted WT1-specific clones only recognized WT1126–134 with low avidities. In contrast, allo-HLA–restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02–positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201–binding peptides was investigated. The self-HLA–restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA–restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201–binding peptides. In conclusion, allogeneic HLA-A*02–restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.

Objective To investigate the presence and patterns of specific IgG subclass recognition by IgM rheumatoid factor (IgM‐RF) and IgA‐RF with a newly developed enzyme‐linked immunosorbent assay (ELISA), which can discriminate between polyspecific and restricted RF responses. Methods Polyspecific and restricted RF responses were determined with our ELISA, which uses individually coated recombinant IgG subclasses instead of polyclonal IgG as target antibodies. Fine specificity was determined using target antibodies with single amino acid mutations in the Fc region. Results In a screening panel of 93 sera that were previously found to be IgM‐RF positive in a conventional RF assay, we were able to discriminate between sera with polyspecific IgM‐RF responses (i.e., RF responses directed against all 4 IgG subclasses) and those with restricted IgM‐RF responses, with low or absent relative reactivity against IgG2, IgG3, or IgG4. We found the largest variation for anti‐IgG3 reactivity. Samples without detectable anti‐IgG4 reactivity formed an independent group from the other restricted RF responses and the polyspecific RF responses. The specificity of these anti‐IgG4–negative sera could be pinpointed to single amino acid differences between IgG1 and IgG4. Polyspecific RF responses more often showed signs of RF response maturation, with more isotype switching to IgA‐RF, as compared to restricted RF responses. In a cohort of IgM‐RF+ and/or anti–citrullinated protein antibody (ACPA)–positive arthralgia patients, we found restricted RF responses in 35% (49 of 140) of RF+/ACPA– patients, while RF+/ACPA+ patients, who have a much higher risk of developing rheumatoid arthritis, virtually always (123 of 128 [96%]) showed a polyspecific RF response. Conclusion IgG subclass–specific RF distinguishes between immature restricted RF responses and potentially more pathogenic, ACPA‐associated polyspecific responses.