BIGReS - Research project

We have no clue about LSR regulation and lack tools for such studies. Dying B-cells escape capture by conventional methods. To break this lock and measure LSR frequency, we will take snapshots of B-cell activation by combining LSR analysis with single-cell detection of IgH defects (with unbalanced IgH/IgL expression), apoptosis blockade and transcription analysis, or by freezing suicidal cells through EBV or LMP2A-mediated rescue. LSR and CSR are competing processes (Fig. 2). CSR is promoted by cytokines and co-stimulatory signals. Our hypothesis is that LSR eliminates sub-optimally stimulated cells, while optimal cytokine-dependent transcription of C genes would promote CSR. We will determine how  LSR relies on the micro-environment, the BCR repertoire and Ag-binding, exploring i) whether LSR specifically targets naive or Ag-experienced switched or unswitched cells ii) the position of LSR in single-cell trajectories of activated B-cells iii) where LSR occurs in lymphoid tissues and its regulation by interactions with stromal and Tfh cells, iv) the  repertoire of LSR-targeted cells, and v) LSR in pathologic conditions…

Figure 2. The IgH locus S regions accessibility is regulated by cytokines and the 3’RR for AID-dependent CSR (left). The 3’RR contains enhancers flanked with LS repeats also targeted by AID in activated cells. AID-dependent LSR results in complete deletion of the IgH C cluster (right).

Beside additive gene therapy, the development of CRISPR/Cas9 genome-editing enables to precisely manipulate genes for a therapeutic effect. This opens new perspectives for immunotherapy with chimeric antigen receptor (CAR) T cells... Compared to T-cells, B-cells are not cytotoxic but they carry the unique property of differentiating into long-lived plasma cells (PCs), able to secrete huge amounts of proteins of interest such as cytotoxic Abs. They could thus also provide next-generation cell therapy tools, and several preliminary studies proposed to re-engineer B-cell specificity and/or to use plasma cells for secretion of a protein of interest. However, technological locks have delayed these developments including high apoptosis rates of B-cells ex vivo, low transfectability, short-term ability to survive in culture, sensitivity to protein misfolding, and susceptibility to cell transformation (about 90% of lymphoproliferatiions are from B-cells) after mutation, recombination or EBV reactivation. 

We and others have now defined conditions with significant expression of recombinant proteins in B cells. Conditions for long-term in vitro proliferation, terminal differentiation and/or maintenance of human B-cells starting from either naïve or memory B-cells were defined (14–16). New methods for precise insertion of genes at defined genomic positions are also available and preliminary tested in human cells. While remodeling of Ig loci by homologous recombination has been done, notably by us, at multiple sites in ES cells, and has provided detailed analysis of how these genes can be reengineered (17–19), we are at the best possible time for exploring how Ig genes can be reformatted in B-cells and how such edited cells can further survive and contribute to immune responses, notably against tumors. 

Applications for immunotherapy are enormous. Current passive protocols targeting tumor Ags or immune checkpoint receptors are indeed highly efficient and increasingly used, but at high costs, especially when treatments need to be prolonged for years. CAR-T-cells also stand as a holy grail but come with severe side effects and limitations (suppression, exhaustion, poor access to some tumor sites…). We thus do believe that reengineered B-cells might cover unmet needs for cancer therapy, by allowing long-term adoptive response with permanent production of the therapeutic Ig, possibility to simultaneously target multiple epitopes of a tumor antigen (or of co expressed tumor antigens with multi-specific Abs), potential ability to re-write immune memory and induce long-lived memory B-cells able to reactivate in case of tumor relapse... Aim 3 is devoted to assembling this puzzle, breaking technological locks and setting up robust protocols for ex vivo generation of “Bespoken-antibody-releasing B-lineage” (BAR-B) human B-cells able to be grafted to recipients after gene editing and then to survive for years in lymphoid tissues as memory cells and/or Ab factories.

A new type of IgG with a glycosylated CH3 (left) and the cell producing it (right)

Maturation of humoral immunity is paint as relying on Activation-Induced cytidine Deaminase (AID) for immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM) of Ig variable genes. This yields sharpened B-cells with a high affinity B-cell receptor (BCR). A wealth of knowledge has accumulated about such long-lived champions able to yield protective antibodies and about conventional CSR/SHM. However, the main outcome of activation in lymphoid tissues is B-cell death, and while both specific antigen (Ag) binding and unspecific innate signals trigger B-cell activation, elimination of cells with unfit BCRs is instrumental in giving way to the B-cell champions. My group and I demonstrated that AID can also delete BCR genes through locus suicide recombination (LSR) which joins Ig heavy chain (IgH) Sµ to like-switch (LS) regions and kills activated B-cells. Since hindered by the rapid death that follows BCR loss, study of AID contribution to B-cell censoring yet remains in its infancy. By exploring the role of LS regions, which are especially structured and expanded in humans, we preliminarily observed another singular event, “enhancer suicide recombination” (ESR) which deletes an IgH superenhancer and likely dampens IgH expression. Exploring these uncharted territories in humans and analyzing mechanisms at work during the elusive stage of activation-induced cell death, where rapidly swept-off dead cells escape analysis, is highly challenging. It requires conceiving highly sensitive methods characterizing LSR, ESR, and variant Ig production and to study B-cells in vitro at the single-cell level, while sophisticated mouse models might bring complementary data in vivo.

In collaboration with Team 1, we will also study with pertinent mouse models the impact of abnormal genetic events on B-cell lymphomagenesis.

Finally, we will work to design new strategies for reshaping the human B-cell genome, with the objective to propose bespoken genetically edited B-cells for therapeutic applications.

By following such unconventional paths, the BIGRES team will try to bring new paradigms and address major issues pertinent to human immunology, immunopathology and immunotherapy.

Health is all about SELECTING the best cells to ensure efficient and safe immunity, which requires electing and training elite soldiers, but also cleaning the deck for them, getting rid of less fortunate competitors. In addition to its acknowledged role in sharpening high-affinity BCRs, a “dark side” of AID action lies in killing or blunting some B-cells.

Knowledge has accumulated about the tip of the iceberg, i.e. the positive selection of initially rare cells, finally predominating. By focusing on the furtive B-cell activation-induced cell death (AICD) stage, we will explore the unbiased landscape of rearrangements mediated by AID before negative selection and cell death. This should score the contribution of LSR to AICD and document still undescribed rearrangements with a potential immunoregulatory role.

Ag-activated B-cells undergo AID-dependent reshaping of Ig genes. SHM within germinal centres (GC) yields cells with higher affinity variable (V) domains, further selected for survival and differentiation. CSR diversifies IgH classes by joining repetitive IgH switch (S) regions. In GCs, negative selection of the less efficient or inappropriately activated B-cells could be accomplished through pathways leading to anergy, death-by-neglect, or AICD.

Since switched antibodies (Abs) are potent actors of immunopathology, means for restricting CSR and reentry of switched cells into SHM are necessary to keep immune responses specific and innocuous. Up to half of GC B-cells die every 6 hours through apoptosis, a majority of them having lost production of functional Ig(1). AID promotes recombination resulting into AICD and we described AID-induced IgH deletion featuring “locus suicide recombination”

(LSR) in the mouse(2). LSR joins Sµ to the 3’ regulatory region (3’RR) located downstream of the locus. The 3’RR super-enhancer promotes SHM and CSR but also includes abundant repetitive DNA arrays resembling S-regions albeit shorter(3–6). As for S regions, 3’RR like-switch” (LS) regions also likely promote DSBs resulting in LSR. By abrogating BCR expression, which is critical for cell survival, LSR washes away the corresponding cells and makes it difficult to quantify such a fleeting event. NGS sequencing recently showed that LSR actively occurs in human inflamed lymphoid tissues without remaining detectable in circulating B-cells, implying that it rapidly ends with B-cell death(7). Such studies were hindered until now by methodological locks and it has notably not been possible to set up any experiment at the single-cell level since the sequencing of LSR deletions (LSR-seq) first needs nested PCR over large distances.

In addition to its higher pertinence for human health, the human B-cell model might be the most adapted for studying LSR: human 3’RRs have much better defined LS repeats than mouse 3'RR, with length and density of repeats very similar to S regions (Fig 1).

Figure 1: Dot plot analysis (YASS algorithm http://bioinfo.lifl.fr/yass/yass.php) of mouse vs human Sa, IgH Ca genes and flanking 3’RR. Green squares mark positions of tandem repeats.

The project addresses several aims. The first one explores singular genetic events affecting human B-cell function and fate. Within the mammalian genome, antigen receptor genes have the unique capacity to undergo programmed gene recombination and/or hypermutation events. B-cell development and activation is thus classically considered to be driven by 3 such processes: V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM). Using various methods, we additionally identified in human B-cells several types of unconventional remodeling events of Ig loci, the regulation of which deserves to be elucidated. Such studies involve the characterization of specific DNA target regions

Researchers: Michel Cogné (PUPH, under IUF delegation for research), Roch Houot (PUPH), Brice Laffleur (CRCN INSERM), Grégory Noel (CR EFS),

Staff: Laurent Deleurme (AI UR1, cell cytometry), Yannic Danger, (¼ time, immunochemistry) and unit core-lab: Simon Leonard (IR INSERM, single-cell), Florence Jouan (Tech UR1, mouse models)

Post-doc: Zeinab Dalloul, Iman Dalloul

3 PhD (Natsuko Ueda, Marine Cahen, Ophélie Dézé)

i) Teams 1 & 2

ii) Members from UMR CNRS 7276, Limoges (JC. Aldigier, L. Delpy, Y. Denizot, J. Feuillard, S. Le Noir, J. Moreau, E. Pinaud)

iii) A Melnick (Cornell University, New York) 

iv) N Fazilleau (CPTP, Toulouse)

v) A. Galy (Généthon , Ivry v) J. Moreaux (IGH, Montpellier)

i) Labellisation ARC: 350k€, 2017-2020 (PI: M. Cogné);

ii) ANR IgH MemImpact: 360k€, 2016-2020 (PI: M. Cogné);

iii) INCa Tfh: 250k€, 2019-2021 (co-PI: M. Cogné),

iv) LLS foundation: 70k$, 2019-2021 (PI: K Tarte)

The unit (UMR INSERM 1236) and the Institute (SFR BIOSIT) will provide the scientific and technical environment necessary to carry out this project. Rennes University has common core facilities also supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and Etablissement Français du Sang (EFS). This brings high-tech equipment and staff in facilities including: animal facility, cytometry and cell sorting, cell and tissue imaging, NGS (ION Proton, GS, Illumina), Transcriptomics, single-cell analysis with 10X or Fluidigm and microfluidic technology for quantitative PCR (Biomark), Biomathematics/informatics facility, and proteomics. The Rennes campus gathers multiple teams in genetics and immunology, organizes meetings and receives visiting scientists, so that the project will receive all possible criticisms from other scientists.