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Direct observation correlates NFκB cRel in B cells with activating and terminating their proliferative program
Haripriya Vaidehi Narayanan1,2, Mark Y Xiang1,2, Yijia Chen1,2
1Signaling Systems Laboratory, Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095.
Insights
Natural variations in NFκB cRel levels control B cell proliferation heterogeneity. High cRel expression initially boosts B cell expansion but leads to earlier decay, revealing a complex regulatory mechanism.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Antibody responses depend on B cell proliferation, which is regulated by affinity-dependent signals.
- B cell proliferation exhibits significant heterogeneity, with individual cells undergoing varying numbers of divisions.
- NFκB cRel is a key transcription factor required for B cell proliferation following immune stimulation.
Purpose of the Study:
- To investigate whether natural variations in NFκB cRel abundance influence the heterogeneity of B cell proliferation.
- To quantitatively analyze the relationship between cRel expression levels and B cell proliferative capacity.
Main Methods:
- Development of a fluorescent reporter (mTFP1-cRel) for real-time observation of cRel in live B cells.
- Live-cell fluorescence microscopy to track cRel distribution and B cell division.
- Mathematical modeling of the NFκB signaling network, including autoregulation and negative feedback loops.
- Analysis of mouse knockouts to validate model predictions.
Main Results:
- cRel is heterogeneously distributed in naïve B cells, with a heavy-tailed distribution favoring high expressors.
- High cRel expressors initiate proliferation faster but exhibit earlier decay in population expansion.
- Mathematical modeling and experimental data confirm that cRel heterogeneity results from a balance between positive autoregulation and negative feedback by IκBε.
- Increased cRel primes B cells for early proliferation via cMyc, but peak induction is constrained by incoherent feedforward regulation, creating a non-monotonic relationship between cRel and proliferation extent.
Conclusions:
- Natural variations in NFκB cRel abundance are a key driver of B cell proliferative heterogeneity.
- The interplay between cRel autoregulation, IκBε inhibition, and cMyc regulation shapes the quantitative dynamics of B cell expansion.
- Direct observation studies are crucial for understanding quantitative biological relationships beyond gene knockout analyses.
Abstract:
Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.
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