Enhanced Antibody Responses in CD19-Cre mice

Published in Protocols & Methods and Immunology
Enhanced Antibody Responses in CD19-Cre mice
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Behind the Paper

The process of scientific discovery is often celebrated for its breakthroughs and transformative insights, but beneath the surface lies an equally important narrative: the meticulous pursuit of rigor, reproducibility and transparency. Our recent study on the widely used CD19-Cre mouse model, published in Scientific Reports, embodies this principle, validating previous findings and underscoring the need for careful scrutiny in experimental systems. CD19, a coreceptor critical to B cell antigen receptor (BCR) signalling, is rendered heterozygous in this model, leading to its diminished expression. Despite the popularity of the CD19-Cre strain, the implications of the reduced CD19 expression in this model had not been fully characterized.

A Tool with Hidden Complexities

This study has its roots in the challenges I faced during my PhD research, which initially focused on the role of a Rab GTPase in B cells. To achieve targeted deletion of this protein, we utilised the CD19-Cre mouse model. Although other models exist (e.g., CD21-Cre, mb1-Cre), we opted for the CD19-Cre model as it was readily available in-house. CD19-Cre mice have long been a cornerstone of B cell-specific genetic studies, enabling targeted manipulation through the Cre-lox system. When setting up my experiments, I sought advice on breeding strategies and controls and was reassured that CD19-WT littermates from the CD19-Cre /Rab-flox would suffice. After all, this model had been used extensively in the field without reported issues, or, if reported, they had been mostly unnoticed. Additionally, selecting CD19-Cre littermates as controls allowed us to considerably reduce the number of breeding pairs and ultimately the overall number of animals, in line with the 3Rs principles.

Over the time of two years, I conducted experiments, analysing data and interpreting findings through the lens of Rab protein function. However, at the time when everything else was ready for manuscript submission, we encountered an unexpected result in our last control experiment, that was supposed to formally show that there is no effect of the Cre cassette in our system. It turned out that the phenotype that we thought originated from our floxed gene, was actually caused by the reduced levels of CD19 due to the insertion of the Cre-cassete in this model. Our CD19-Cre mice, compared to CD19-WT mice, exhibited elevated antibody responses to both T-dependent (NP-KLH) and T-independent (NP-Ficoll) immunizations. Basal levels of IgM antibodies were also elevated, and in vitro experiments showed increased B cell class-switch recombination.

This realization marked a turning point. While it was disheartening to realize that the project had taken an unplanned detour, this experience reflects the collective learning process that science often demands. What began as a setback ultimately became an opportunity to address a gap in the literature. Notably, we found a paper published that same year by Zhao et al. also reporting increased antibody responses in CD19-Cre animals upon immunization. Motivated by the opportunity to strengthen these claims, we decided to publish our findings as well.

Together, our studies suggest that reduced CD19 expression may subtly influence B cell function in ways that had previously gone unnoticed. It is possible that the effects of hypomorphic CD19 expression are additionally influenced by the genetic background, explaining for the variability in the reported phenotypes (Zhao et al., 2022).

A Broader Perspective

Our findings underscore the need for heightened awareness when using widely adopted models in research. They also serve as a reminder that even established tools require ongoing scrutiny. Reproducibility is not merely a technical requirement; it is the foundation of scientific trust. As researchers, we have a responsibility to critically evaluate the tools and models we rely on, openly share our findings, and advocate for transparency.

Special recognition goes to reviewers who prioritize the rigor of the science over asking for additional experiments. In our case, we also had received constructive feedback from a reviewer requesting for additional controls, exemplifying the role reviewers play in strengthening the integrity of scientific research. By sharing the story behind this paper, we hope to contribute to a culture where careful validation is the norm, not the exception.

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Immunology
Life Sciences > Biological Sciences > Immunology
Immune Cell Signalling
Life Sciences > Biological Sciences > Immunology > Immune Cell Signalling
Immunological Models
Life Sciences > Biological Sciences > Biological Techniques > Biological Models > Immunological Models

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