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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - BCRdangerCOMPETITION (The role of danger signal and cellular competition in the elimination of B cells lacking the B cell receptor.)

Teaser

Summary

Immune reactions protect our bodies from dangers such as foreign pathogens (bacteria, viruses, fungi), cellular transformation or help during wound healing. Our immune system is able to recognize and fight but also to learn thanks to lymphocytes. B lymhpocytes (B cells) are responsible for antibody production and immunological memory. A B cell carries an antibody structure â€“ a specialized B-cell receptor also called immunoglobulin (Ig) - on its surface.
During a B cell life, there are two time points when the Ig is actively engaged and signals: in early life when Ig is produced and in an immune reaction when Ig engages with an intruder. Between, there is a window of a month or two when B cells are resting.
This evoked curiosity, do B cells need the Ig in a resting state? The laboratory of Prof. Klaus Rajewsky discovered that resting B cells depend on the expression of Ig, without Ig B cells die. A â€œtonic signalâ€, a hypothetical signal coming from the Ig in a resting state has been proposed as essential for the maintenance of B cells. However, attempts to identify the trigger which activates such tonic signal have been vain.
In the BCRdangerCOMPETITION project, we addressed alternative mechanisms which might induce B cell death upon loss of the Ig â€“ a cellular stress response, particularly unfolded protein response (UPR). This is initiated once a cell synthetizes more proteins than it can fold (shape into correct confirmation) and in order to re-establish equilibrium induces the expression of proteins involved in stress response, e.g. facilitators of protein folding. If the stress resolves, the cell survives and turns down the stress response, however, if the stress persists, the prolonged overexpression of proteins involved in UPR leads to cell death. Ig is a complex of multiple proteins that assemble inside the cells and are transferred as Ig complex to the cellular surface. When among the proteins forming the Ig complex we delete one essential part, the other Ig proteins remain expressed but cannot form the Ig complex. In theory, they could accumulate, overwhelm the folding machinery of a cell activating the stress response which kills the B cell. To our surprise and contrary to our hypothesis, B cells without Ig were unable to induce the mentioned stress response. We propose a model where the presence of Ig is required to keep the expression of proteins involved in stress response at physiological levels which maintain the function of cellular organelles and anabolic metabolism. Although counterintuitive at first sight, stress response at physiological levels maintained by the presence of Ig is vital for cellular survival. This finding might uncover a novel survival signal that requires the physical presence of Ig to maintain the stress response functional at physiological levels.
In parallel, we addressed another potential mechanism responsible for B cell death upon loss of the Ig: cellular competition. The phenomenon of cellular competition has recently been identified as the cause of disappearance of Ig-negative lymphoma cells (malignant B cells). While lymphoma cells without Ig can grow normally in isolation, they disappear when placed in competition with Ig-positive lymphoma cells. We addressed the extent of cellular competition in primary B cells using the novel CRISPR/Cas9 mediated mutagenesis of primary B cells. We have set up an efficient protocol to induce Ig deletion in primary B cells and observed them under conditions resembling an immune reaction. B cells without Ig were unable to persist and were outcompeted by their Ig-positive counterparts in only two days. So far, our results show that Ig deletion is detrimental for primary B cells growing under conditions resembling an immune reaction and provide experimental evidence for an old observation, namely, that Ig- B cells cannot be detected after an immune reaction in mice or humans. We are currently working on improving our experimental setup to o

Work performed

I measured the extent of stress response in primary B cells upon chemical stress induction and upon acute Ig deletion. While chemical induction led to an increased stress response and primary B cell apoptosis, stress response was not induced upon Ig ablation in primary B cells (in contrast to one of our hypotheses).
I set up a parallel experimental system of CRISPR/Cas9 mediated Ig ablation in human lymphoma cell lines and, similarly to primary murine B cells, stress response was reduced upon Ig ablation. Electron microscopy analysis of B cells without Ig (Ig- B cells) revealed signs of impaired structures of cellular organelles. Experiments are underway to understand how the damaged organelles impact the metabolism of Ig- B cells. In an ongoing collaboration with the group of J. Jellusova (BIOSS, Freiburg) we identified the dependence of B cells on the physical presence of the Ig that is essential for B cell survival. These results are in preparation for publication.
I assessed the fitness of Ig- B cells obtained by CRISPR-Cas9 mediated mutagenesis of primary B cells in cell culture systems resembling an immune reaction. Ig- B cells showed dramatically decreased fitness as compared to Ig+ B cells.
Results obtained from the BCRdangerCOMPETITION project have been presented to the international research community at the following scientific conferences: Protein quality control (Sant Feliu de Guixols, 2017) and 17th B Lymphocyte Forum (Collegium GlashÃ¼tten, 2019). In addition, a manuscript (in preparation) will be submitted to a pre-print server and then for a peer-reviewed scientific publication. This project has been presented to professionals active in scientific communication and museum exhibitions at ECSITE (Porto, 2017), to a broad public audience at The Long Night of the Sciences (MDC Berlin, 2018) and at the 100.000th MSCA Fellow Awards Ceremony (Brussels, 2017) with the attendance of Tibor Navracsics, Commissioner for Education, Culture, Youth and Sport. My project was featured in online publications and in social media.

Final results

Our research is fundamental for the field of B cell biology and might have future implications for the development of treatment strategies for B cell malignancies. Although most B cell malignancies are Ig-positive, increasing clinical data indicate that the fraction of Ig- lymphomas is not negligible. These lymphomas might arise spontaneously or as a result of a cellular selection in response to treatment targeting Ig-mediated signaling. The biology of Ig-negative B cells is different from that of Ig-positive ones, thus also the treatment strategies for Ig-negative lymphomas must be directed specifically at the weaknesses of Ig-negative B cells.