Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Gabriel D. Victora
Keywords
cell-cell interactions, B cell selection, T cell help, germinal centers, single-cell RNA sequencing (scRNA-seq), uLIPSTIC
Abstract
In multicellular organisms, each cell has a distinct identity characterized by unique transcriptional and chromatin accessibility profiles. However, to truly understand the organization and function of such systems, it is not enough to merely characterize individual components; we must also investigate how these components interact and communicate through direct cellular interactions. This is especially true in the immune system, where cell-cell interactions are fundamental. For instance, the priming of antigen-specific T cells relies on their selective engagement with antigen-presenting cells (APCs) displaying the T cell's cognate antigen, distinguishing them from a pool of non-specific counterparts. Similarly, the production of antibodies by plasma cells depends on B cell receptors capturing antigen for presentation to helper T cells. These processes exemplify the immune system's dependence on its cells' ability to locate their interacting partners and exchange critical information. During infection, the affinity of circulating serum antibodies increases over time in a process known as antibody affinity maturation. This phenomenon is driven by the positive selection of B cells expressing higher-affinity B cell receptors (BCRs) to expand and occurs within specialized structures called germinal centers (GCs). While it is well-established that T-B interactions are essential for the formation and maintenance of GCs, the precise role of T cell help as the primary driver of positive selection remains debated. In this work, I introduce a reingineered method, LIPSTICv2, to address this long-standing question. By integrating interaction-based single-cell transcriptomics, I provide clear evidence that T cell help is preferentially delivered to B cells expressing higher-affinity BCRs. Furthermore, the magnitude of this T cell help dictates the extent of upregulation of transcriptional programs associated with B cell clonal expansion and positive selection. Building on this work, I then developed uLIPSTIC, an enhanced version of LIPSTIC designed to enable quantitative and unbiased analysis of cellular interactions. Whereas earlier iterations of LIPSTIC were confined to interactions mediated by CD40 and CD40L, uLIPSTIC eliminates this limitation by anchoring Sortase A (SrtA) and its oligoglycine acceptor non-specifically to the cell membrane at high density. This innovation allows enzymatic labeling between any membranes that come into close proximity, vastly expanding the range of potential applications. By coupling uLIPSTIC with droplet-based scRNA-seq, quantitative interaction-based transcriptomics is achieved. This approach has two distinct applications: First, in an 'atlas' mode, it allows for the comprehensive mapping of the cellular interactome within a given donor cell population. Second, in a 'mechanistic' mode, uLIPSTIC signal intensity can be correlated with specific gene expression profiles or signatures, enabling the elucidation of molecular pathways underlying specific interactions. This body of work underscores the transformative potential of adding interaction resolution to our understanding of cellular function. Both LIPSTICv2 and uLIPSTIC represent cutting-edge technologies that allow for the detailed study of cellular interactions in vivo, offering new insights into the molecular dynamics that govern immune responses and beyond.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Nakandakari-Higa, Sandra, "Recording Cellular Interactions in Germinal Centers and Beyond" (2025). Student Theses and Dissertations. 819.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/819
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy