Biology subjects

Rethi, B.

Publications and source records attributed to Rethi, B..

3 recordsLinked to original sources

bioRxiv2026

A new humanized TCR transgenic mouse model to study citrullinated tenascin C reactive T cells relevant to rheumatoid arthritis

ObjectivesAutoreactive T cells recognizing citrullinated antigens, although rare and difficult to study, are implicated in rheumatoid arthritis (RA). To allow functional studies and manipulation of such T cells, we have generated and characterized transgenic mice expressing a TCR cloned from an RA patient and reactive with a prominent target, citrullinated tenascin C (citTNC). MethodsA humanized TCR transgenic (hTCR-tg) mouse recognizing the citrullinated TNC22 (citTNC22) antigen in an HLA-DRB1*04:01 (HLA-DR4) restricted manner was developed by genetically engineering a chimeric TCR expressing murine constant domains and human V(D)J sequences. hTCR-tg mice were immune phenotyped in murine H-2b and humanized HLA-DR4 backgrounds using full spectrum flow cytometry, cytokine ELISAs and fluorospot assays at steady-state and after antigen challenge. Additionally, we investigated the presence of antibodies to citTNC and arthritis following citTNC protein immunization. ResultsThymic selection of hTCR T cells differed between the two MHC-II alleles, with a normal CD4+ T cell development observed solely under HLA-DR4 restriction. The chimeric hTCR maintained its citTNC22 specificity in vitro and ex vivo, without cross-reactivity to native TNC22 or other citrullinated autoantigens. hTCR-tg CD4+ T cells responded to antigen challenge in vivo and provided support to IgG class-switch and antigen-specific antibody production. Moreover, protein immunized hTCR-tg mice developed arthritis after periarticular challenge with citTNC. ConclusionshTCR-tg mice expressing an RA patient-derived autoreactive TCR develop functional antigen-specific and HLA-restricted CD4+ T cells. The combination of humanized HLA-DR4 and TNC22 mice will be a valuable tool in the development of antigen-specific therapies translatable to human disease.

immunology↗

bioRxiv2025

Engineered Fab glycosylation of a blood-brain barrier transporter single-chain variable fragment preserved functionality but did not impact anti-drug antibody formation

The single-chain variable fragment (scFv) of the 8D3 antibody enables blood-brain barrier transport via transferrin receptor-mediated transcytosis. However, when fused to therapeutic antibodies, scFv8D3 increased immunogenicity and triggered anti-drug antibodies (ADA). Sialylation of N-linked glycans can critically influence protein immunogenicity, potentially conferring immunosuppressive properties and extended half-life. We investigated whether introducing sialic acid residues to scFv8D3 through engineered fragment antigen-binding (Fab) glycosylation could reduce ADA formation. We generated two novel variants with distinct N-linked glycosylation motifs (NXT/S), which both displayed sialic acid residues and retained functionality, as evidenced by the preservation of transferrin receptor binding and brain uptake. However, Fab glycosylation did not significantly alter pharmacokinetics or ADA production in mice. Our findings demonstrated that introducing Fab glycosylation is feasible, without compromising biological activity. Nonetheless, this strategy alone was insufficient to mitigate immunogenicity, underscoring the need for alternative approaches to improve the long-term safety of brain-targeting therapeutics. Statement of significanceSialylation of N-linked glycans can critically influence protein immunogenicity. We introduced N-linked Fab glycosylation sites into a scFv-based therapeutic construct with the aim of reducing ADA production upon repeated administration to mice. Although the sialylated variants retained functionality, pharmacokinetics and ADA levels remained unaffected, suggesting limited immunomodulation via engineered sialylation.

bioengineering↗

bioRxiv2022

Divergent and dominant anti-inflammatory effects of patient-derived anti-citrullinated protein antibodies (ACPA) in arthritis development

About two thirds of rheumatoid arthritis (RA) patients develop anti-citrullinated protein antibodies (ACPA), which are a characteristic of the disease and sparsely observed in other clinical settings or in the healthy population. ACPA are used as a diagnostic criterion and often develop prior to diagnosis. Therefore, ACPA titers are important in the identification of individuals at risk of developing RA. Interestingly, the titers and target cross-reactivity of ACPA increase by time of diagnosis, suggesting a causality between anti-citrulline reactivity and the development of RA. However, only 50% of ACPA-positive at-risk individuals will progress to a clinical RA diagnosis. This observation suggests that there are different types of ACPA and that their relationship with RA development is more complex than presently assumed. To address the biological effect of ACPA in the establishment and development of inflammatory arthritis, we made use of the collagen antibody-induced arthritis (CAIA) model of passive arthritis. Using seven unique patient derived monoclonal ACPA, we observed that ACPA are predominantly anti-inflammatory, with some clones (C03 and BVCA1) completely inhibiting disease development. We have also identified a clone (C04) as having a disease-prone effect, by increasing and sustaining disease prevalence. This clone had previously been reported as being pro-inflammatory in a different model of joint inflammation. The anti-inflammatory effects of C03 were dependent on Fc{gamma}R, since neither F(ab)2 or a mutated Fc-null GRLR-C03 clone could mediate disease protection. Most importantly, mice receiving polyclonal ACPA enriched from RA sera were also partially protected from disease. Looking at the cell and tissue of origin of the ACPA clones, their V(D)J sequences, Fc glycosylation pattern and fine-specificities, we could not identify a common feature between anti-inflammatory ACPA distinguishing them from those without the properties. Together, our data demonstrates that circulating ACPA in RA patients are predominantly anti-inflammatory, emphasizing the need to study ACPA repertoires in order to determine their influence on the clinical outcome of the patient or at-risk individual.

immunology↗