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T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette ("scFull-Ig") that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further pave the way for future B cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions., (© 2024. The Author(s).)

Introduction: Follicular Lymphoma (FL) results from the malignant transformation of germinal center (GC) B cells. FL B cells display recurrent and diverse genetic alterations, some of them favoring their direct interaction with their cell microenvironment, including follicular helper T cells (Tfh). Although FL-Tfh key role is well-documented, the impact of their regulatory counterpart, the follicular regulatory T cell (Tfr) compartment, is still sparse., Methods: The aim of this study was to characterize FL-Tfr phenotype by cytometry, gene expression profile, FL-Tfr origin by transcriptomic analysis, and functionality by in vitro assays., Results: CD4 + CXCR5 + CD25 hi ICOS + FL-Tfr displayed a regulatory program that is close to classical regulatory T cell (Treg) program, at the transcriptomic and methylome levels. Accordingly, Tfr imprinting stigmata were found on FL-Tfh and FL-B cells, compared to their physiological counterparts. In addition, FL-Tfr co-culture with autologous FL-Tfh or cytotoxic FL-CD8 + T cells inhibited their proliferation in vitro . Finally, although FL-Tfr shared many characteristics with Treg, TCR sequencing analyses demonstrated that part of them derived from precursors shared with FL-Tfh., Discussion: Altogether, these findings uncover the role and origin of a Tfr subset in FL niche and may be useful for lymphomagenesis knowledge and therapeutic management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rodriguez, Alizadeh, Lamaison, Saintamand, Monvoisin, Jean, Deleurme, Martin-Subero, Pangault, Cogné, Amé-Thomas and Tarte.)

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as aggregates of tumor cells densely packed with their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. In vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B-cell lymphomas remain scarce, while all these parameters are key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on cell encapsulation inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D model incorporating lymphoma B cells, extracellular matrix (ECM), and/or tonsil stromal cells (TSC). Under 3D confinement, lymphoma B cells were able to form cohesive spheroids resulting from overexpression of ECM components. Moreover, lymphoma B cells and TSC dynamically formed self-organized 3D spheroids favoring tumor cell growth. 3D culture induced resistance to the classical chemotherapeutic agent doxorubicin, but not to the BCL2 inhibitor ABT-199, identifying this approach as a relevant in vitro model to assess the activity of therapeutic agents in B-NHL. RNA-sequence analysis highlighted the synergy of 3D, ECM, and TSC in upregulating similar pathways in malignant B cells in vitro than those overexpressed in primary lymphoma B cells in situ. Finally, our 3D model including ECM and TSC allowed long-term in vitro survival of primary follicular lymphoma B cells. In conclusion, we propose a new high-throughput 3D model mimicking lymphoma tumor niche and making it possible to study the dynamic relationship between lymphoma B cells and their microenvironment and to screen new anti-cancer drugs., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Fibroblastic reticular cells (FRCs) are the specialized lymphoid stromal cells initially identified as triggering T-cell recruitment and dynamic motion in secondary lymphoid organs. Interestingly, FRCs also display antigen presentation capacities and support lymphocyte survival. CXCR5 + CD4 + follicular T cells are important players of B-cell maturation and antibody response. Our study reported that in vitro -differentiated FRC-like cells enhanced the growth of the whole CXCR5 + CD4 + T-cell compartment, while enhancing IL-4 secretion specifically by the PD1 dim CXCR5 + CD4 + cell subset, in a Notch- and ICAM1/LFA1-dependent manner. In addition, we revealed that in follicular lymphoma (FL) tissues, previously identified as enriched for PD1 hi CXCR5 hi CD4 + mature follicular helper T cells, PD1 dim CXCR5 + CD4 + T cells displayed an enrichment for Notch and integrin gene signatures, and a Notch and ICAM-1-dependent overexpression of IL-4 compared to their non-malignant counterparts. These findings suggest that the crosstalk between FRCs and CXCR5 + PD1 dim CD4 + T cells may contribute to the FL IL-4 rich environment, thus providing new insights in FL lymphomagenesis., (Copyright © 2020 Misiak, Jean, Rodriguez, Deleurme, Lamy, Tarte and Amé-Thomas.)