OP0044 ANALYSIS OF IMMUNOGLOBULIN REPERTOIRE REVEALS ALTERED PERIPHERAL B CELL TOLERANCE CHECKPOINTS IN PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS
Authors: E. Marasco, A. Aquilani, I. Caiello, R. Nicolai, G. Tarantino, S. Magni-Manzoni, R. Carsetti, F. De Benedetti
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Juvenile Idiopathic Arthritis (JIA) is the most common form of chronic arthritis in childhood. The adaptive branch of the immune system plays a crucial role in the development of JIA. We have showed that switched memory B cells are expanded in patients with oligoarticular- and polyarticular-JIA. <h3>Objectives:</h3> To characterize the antigen-experienced immunoglobulin repertoire, we performed deep sequencing of the switched repertoire in peripheral blood (PB) of oligo-/ploy-JIA patients(n=14) and age matched controls(n=4), and in matched synovial fluid (SF) of oligo-/ploy-JIA patients(n=7). <h3>Methods:</h3> We employed a cDNA-based-5′RACE approach with unique molecular identifiers. De-multiplexing, UMI extraction, and UMI-based consensus assembling were performed using the MIGEC software. Clonotype assembly were performed using MiXCR. SHM analysis was performed with SHazaM. B cell tolerance checkpoints were analyzed with a validated flow cytometry-based system (Malkiel S, Arthritis Rheumatol. 2016). <h3>Results:</h3> Immunoglobulin heavy chain variable gene segment usage frequency was similar between patients and controls, and PB and SF. Thus, we did not observe clustering of patients from controls. Analysis of averaged complementarity-determining region 3 (CDR3) repertoire characteristics revealed that CDR3 length was similar among the three groups, whereas physico-chemical characteristics of amino acid residues were significantly different in the IgG repertoire, but not in the IgA repertoire. IgG from SF showed higher charge and polarity and an increased hydrophobicity index compared to PB of controls and patients. We then analyzed the frequency of somatic hypermutation (SHM) and the selection pressure with SHazaM. We found a significant reduction in the frequency of SHM and a higher frequency of sequences with less than 10 mutations per Ig molecule in patients (in both PB and SF) than controls. The IgG repertoire in SF exhibited decreased positive selection in CDRs. To further characterize the immunoglobulin repertoire, we performed a flow cytometry-based staining to assess the frequency of autoreactive B cells in the transitional, naïve and memory B cell subsets in PB and also of memory B cells in SF. We found that central tolerance checkpoints are effective in both JIA patients and controls: the frequency of autoreactive B cells decreased from transitional, to naïve B cells in both JIA patients and controls. However, the frequency of autoreactive B cells was higher in memory B cells of JIA patients compared to controls, with the highest frequency observed in memory B cells of SF. <h3>Conclusion:</h3> Altogether, the observed changes in the IgG repertoire in PB and SF establish an altered peripheral selection process of IgG+ B cells in oligo-/ploy-JIA with a decreased load of SHM. Our data show for the first time that an altered GC response, leading to the accumulation of autoreactive B cells in the memory compartment, is a feature of oligo-/ploy-JIA. <h3>REFERENCES:</h3> <b>NIL.</b> <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> Emiliano Marasco: None declared, Angela Aquilani: None declared, Ivan Caiello: None declared, Rebecca Nicolai: None declared, Giusyda Tarantino: None declared, Silvia Magni-Manzoni: None declared, Rita Carsetti: None declared, Fabrizio De Benedetti Novartis, SOBI.