Upon T cell receptor (TCR) engagement, phosphorylation of the LAT adapter protein enables binding of the enzyme PLC-γ1 to a Gads/SLP-76 dimer, forming a tetrameric structure. The interaction between SLP-76 and PLC-γ1 is weak within this heterotetramer, and the relevant binding sites of SLP-76 and PLC-γ1 are highly conserved in vertebrates. We generated a mouse with a T cell-specific mutation in the SLP-76 that enhanced its affinity for PLC-γ1, thereby increasing PLC-γ1 activity and TCR signal strength. This mutation not only altered the development of αβTCR thymocytes, invariant NKT cells, and intraepithelial lymphocyte precursors but also impaired the generation of central memory CD8+ T cells upon acute viral infection and the humoral immune response mediated by germinal center T follicular helper T cells. These findings suggest that the conserved weak SLP-76/PLC-γ1 interaction is important for the controlled activation of PLC-γ1, thus fine-tuning TCR signal strength to optimize T cell-mediated immunity.
© 2026. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

The adult B cell pool is a mosaic comprising short-lived naive B cells and long-lived memory. Using genetic time stamping, we have previously shown that early-life-origin (ELO) B cells contribute substantially to the adult mouse immune system. Here, we show that they share a memory-like signature, with ELO B-1 cells being enriched for the PD-L2/CD80 double-positive (DP) immunophenotype. Indeed, microbial antigen exposure in neonates expands distinct specificities within the DP B-1 cell compartment, identifying it as a reservoir of immunoglobulin (Ig)M memory. B cell chronic lymphocytic leukemia (CLL) is a disease marked by the accumulation of memory-like cells. By applying time stamping to a mouse model of unmutated CLL, we demonstrate that leukemic expansion is driven by B-1 clones that arise prior to postnatal day 10. Importantly, B-1 cells in mice and humans share molecular features with unmutated CLL, altogether supporting a potential contribution of ELO B cells to this disease.
Copyright © 2026 The Author(s). Published by Elsevier Inc. All rights reserved.

Adipose tissue immune cells are heterogeneous and dynamic, alter metabolism, and drive immune responses. Here, we present a protocol for assessment and characterization of murine adipose tissue immune cells using fluorescence-based flow cytometry and sorting into pure populations. We describe steps for isolation of the stromovascular fraction, antibody staining, and data collection by flow cytometry. We also discuss common issues and troubleshooting steps. For complete details on the use and execution of this protocol, please refer to Carey et al.1.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.
© 2023 The Authors. Published by American Chemical Society.

Vaccines typically protect against (re)infections by generating pathogen-neutralising antibodies. However, as we age, antibody-secreting cell formation and vaccine-induced antibody titres are reduced. Antibody-secreting plasma cells differentiate from B cells either early post-vaccination through the extrafollicular response or from the germinal centre (GC) reaction, which generates long-lived antibody-secreting cells. As the formation of both the extrafollicular antibody response and the GC requires the interaction of multiple cell types, the impaired antibody response in ageing could be caused by B cell intrinsic or extrinsic factors, or a combination of the two. Here, we show that B cells from older people do not have intrinsic defects in their proliferation and differentiation into antibody-secreting cells in vitro compared to those from the younger donors. However, adoptive transfer of B cells from aged mice to young recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the GC during the early stages of GC formation. In contrast, by the peak of the GC response, GC B cells derived from the donor cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Collectively, this study shows that B cells from aged individuals are not intrinsically defective in responding to stimulation and becoming antibody-secreting cells, implicating B cell-extrinsic factors as the primary cause of age-associated impairment in the humoral immunity.
© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.