Spleen Study Reveals Proliferation, Not Receptor Sequence, Drives T Cell Repertoire Layout

The adaptive immune system faces a staggering task: with a repertoire of T cell receptors theoretically capable of recognizing billions of distinct molecular shapes, it must somehow organize its cellular forces so that the right cells are in the right place at the right time. For decades, immunologists have wondered whether the sequence of a T cell receptor itself influences where that cell resides within lymphoid organs, or whether the apparent spatial patterns of receptor repertoires are simply byproducts of anatomy and cell division. A new computational and experimental study in mice, published in PLOS Computational Biology, comes down firmly on the side of the latter, and in doing so delivers a cautionary message for anyone interpreting spatial immune data.

A team led by Franka Buytenhuijs, Cornelia Tune, Thomas C. Smits, Andrea Schampel, Lisa-Kristin Schierloh, René Pagel, Jürgen Westermann, and Johannes Textor set out to dissect how the T cell receptor repertoire is distributed across the major compartments of the mouse spleen. The spleen is an ideal testing ground for this question because it contains clearly demarcated regions: the T cell zones, where T cells normally congregate; the B cell zones, which are dominated by B lymphocytes but normally contain a small number of T cells; and the germinal centers, specialized structures within B cell zones where antibody affinity maturation takes place and where T helper cells provide critical support to B cells.

The researchers’ strategy relied on precision microdissection. Rather than analyzing whole spleens, they physically separated T cell zones, B cell zones, and germinal centers from both naïve mice and mice that had been immunized with a complex antigen. From each compartment they extracted DNA and sequenced the complementarity-determining region 3 of the T cell receptor beta chain, abbreviated CDR3β. This short stretch of amino acids is the most variable part of the receptor and serves as a practical fingerprint of clonal identity, allowing the team to track individual T cell clones and measure how abundant each clone was in each splenic neighborhood.

The initial comparison produced a striking result: the receptor repertoires of the three compartments looked substantially different from one another. At face value, this seemed to support the idea that receptor-intrinsic features guide T cell localization. But the team did not stop at the descriptive level. They built statistical models that asked a more demanding question: could the observed differences be explained without invoking any special homing properties of particular receptors, simply by accounting for how big each compartment is and how fast the cells within it are dividing?

The answer, in most cases, was yes. During the steady state, before any immune challenge, the handful of T cells found in B cell zones turned out to strongly resemble random subsets of the T cell zone repertoire. In other words, the T cells that stray into B cell territory appear to be an unbiased sample of the broader T cell population rather than a select subset with distinctive receptors. Once the effect of compartment size was factored into the analysis, the apparent compositional differences between T cell zones and B cell zones largely evaporated.

Immunization changed the picture in a dynamic and informative way. After the mice were challenged with antigen, expanded clones began to appear in the T cell zones by day three, marking the onset of the proliferative response. As the response progressed, these expanded clones later shifted their dominance toward the B cell zones, consistent with the migration of activated T helper cells to the sites where they assist B cells. Germinal centers harbored clones that had expanded even further, and these clones showed a tendency to group into clusters with high sequence similarity, reflecting the family relationships of proliferating T cells that had descended from common precursors.

To make sense of these temporal patterns, the researchers turned to a classical mathematical framework: the Galton-Watson branching process, a stochastic model of population growth in which each cell independently divides or fails to divide according to fixed probabilities. When they fitted this model of T cell proliferation to their data, it explained the repertoire differences observed during the immune response remarkably well. The implication is provocative: the central factor shaping the spatial differences in the splenic T cell receptor repertoire may simply be how much the cells in each compartment have proliferated, not any receptor-sequence-dependent trafficking rule.

The team also examined whether specific structural features of the receptor, beyond clonal identity, correlated with compartmental localization. Two features stood out initially: the length of the CDR3β region and the closeness of the receptor sequence to its germline configuration, a measure of how much the receptor has diverged from the inherited template. Both features showed more pronounced differences between compartments in the raw data, hinting that they might influence localization. Yet here again the models of random distribution and proliferation accounted for these differences almost entirely. Apparent sequence-based patterns dissolved once the confounding effects of compartment size and clonal expansion were explicitly modeled.

Taken together, the findings suggest that splenic T cell receptor repertoires are shaped predominantly by compartment size and proliferation rates rather than by T cell receptor sequence features. This conclusion carries practical consequences for the rapidly growing field of spatial immune profiling. As technologies for sequencing receptors from anatomically defined regions proliferate, researchers increasingly report spatial differences in repertoire composition and interpret them as evidence of receptor-guided positioning. This study demonstrates that such interpretations are unsafe without quantitative controls: a repertoire difference between two compartments can arise purely because one compartment is small, or because the cells within it have undergone more rounds of division, without any receptor-intrinsic homing behavior at all.

The authors argue that a quantitative assessment of T cell receptor repertoire dynamics across heterogeneous spatial compartments during an immune response is only possible when the effects of compartment size and proliferation are explicitly accounted for. Their work provides a template for how to do this, combining microdissection, high-throughput CDR3β sequencing, and mechanistic models of clonal expansion. For immunologists studying how the body marshals its T cell forces during infection or vaccination, the message is clear: before attributing spatial repertoire patterns to the sequences of the receptors themselves, one must first ask whether simple arithmetic of anatomy and cell division can explain the observation. In the mouse spleen, at least, it largely can.

Subject of Research: Spatial distribution of the splenic T cell receptor repertoire during immune responses in mice

Article Title: How compartmentalization and proliferation shape the splenic T cell repertoire during an immune response to a complex antigen in mice

Article References: Buytenhuijs, F., Tune, C., Smits, T. C., Schampel, A., Schierloh, L.-K., Pagel, R., Westermann, J., & Textor, J. (2026). How compartmentalization and proliferation shape the splenic T cell repertoire during an immune response to a complex antigen in mice. PLOS Computational Biology, 22(10), e1014859. https://doi.org/10.1371/journal.pcbi.1014859

Image Credits: AI Generated

DOI: 10.1371/journal.pcbi.1014859

Keywords: T cell receptor, repertoire sequencing, spleen, germinal centers, compartmentalization, clonal proliferation, Galton-Watson model, CDR3β, immunization, computational biology, spatial immunology, mouse model