Dominik Schienstock

Exhausted CD8⁺ T (T_EX) cell responses are maintained by precursors of exhausted T (T_PEX) cells that possess high self-renewal and developmental potential. T_PEX cells also drive the proliferative burst of effector T cells upon therapeutic immune checkpoint blockade (ICB). However, the spatial context and signals that regulate their differentiation and function are not well defined. Here we identify developmental and functional compartmentalization of T_PEX and T_EX cells across secondary lymphoid organs during chronic infection. We show that stem-like CD62L⁺ T_PEX and effector-like CX3CR1⁺ T_EX cells constitute a distinct developmental lineage that is promoted by the lymph node (LN) microenvironment and dependent on the transcription factor KLF2. LNs act as a niche in which migratory dendritic cells provide antigen and costimulatory signals to maintain the proliferative fitness of CD62L⁺ T_PEX cells and generation of CX3CR1⁺ T_EX cells. Moreover, LNs exclusively drive the proliferative burst and systemic dissemination of CX3CR1⁺ T_EX cells during ICB. Thus, our findings identify a unique role for LNs in the maintenance of T cell differentiation and function during systemic chronic infection and ICB therapy.

Fibroblastic reticular cells (FRCs) are specialized fibroblasts that construct secondary lymphoid organs where they provide crucial signals for immune cell homeostasis and migration. While splenic FRCs are thought to support antiviral T cell responses, their role remains unclear. Here, we found that ablation of splenic FRCs impaired virus-specific CD8 ⁺ T cell responses during lymphocytic choriomeningitis virus (LCMV) infection. Immunofluorescence imaging revealed that FRCs promote CD8 ⁺ T cell clustering with type 1 conventional dendritic cells (cDC1) in the T cell zone before migration to the infected marginal zone. Without FRCs, T cells instead clustered with cDC1 and virus-infected cells in the marginal zone, leading to suboptimal priming. Mechanistically, FRCs coordinated early viral replication and the inflammatory milieu for optimal DC activation, and an intact FRC network was crucial for generating effector T cells and maintaining protective memory T cells. Thus, splenic FRCs provide essential lymphoid niches for antiviral T cell responses.

With the ever-increasing complexity of microscopy modalities, it is imperative to have computational workflows that enable researchers to process and perform in-depth quantitative analysis of the resulting images. However, workflows that allow flexible, interactive and intuitive analysis from raw images to analysed data are lacking for many experimental use-cases. Notably, integrated software solutions for analysis of complex 3D and live cell images are sorely needed. To address this, we present Cecelia, a toolbox that integrates various open-source packages into a coherent data management suite to make quantitative multidimensional image analysis accessible for non-specialists. We describe the application of Cecelia to several immunologically relevant scenarios and the development of an unbiased approach to distinguish dynamic cell behaviours from live imaging data. Cecelia is available as a software package with a Shiny app interface (https://github.com/schienstockd/cecelia). We envision that this framework and its approaches will be of broad use for biological researchers.

With the ever-increasing complexity of microscopy modalities, it is imperative to have computational workflows that allow researchers to process and perform in-depth quantitative analysis of the resulting images. However, workflows that enable flexible, interactive and intuitive analysis from raw images to analysed data are lacking. We present Cecelia, a toolbox that integrates various open-source packages into a coherent data management suite to make quantitative image analysis accessible for non-specialists.

A diverse fibroblastic stromal cell landscape in the spleen directs tissue homeostasis and immunity.

The analysis of cellular behavior using intravital multi‐photon microscopy has contributed substantially to our understanding of the priming and effector phases of immune responses. Yet, many questions remain unanswered and unexplored. Though advancements in intravital imaging techniques and animal models continue to drive new discoveries, continued improvements in analysis methods are needed to extract detailed information about cellular behavior. Focusing on dendritic cell (DC) and T cell interactions as an exemplar, here we discuss key limitations for intravital imaging studies and review and explore alternative approaches to quantify immune cell behavior. We touch upon current developments in deep learning models, as well as established methods from unrelated fields such as ecology to detect and track objects over time. As developments in open‐source software make it possible to process and interactively view larger datasets, the challenge for the field will be to determine how best to combine intravital imaging with multi‐parameter imaging of larger tissue regions to discover new facets of leukocyte dynamics and how these contribute to immune responses.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4⁺ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKR^KI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKR^KI/KI spleen, with the remaining MHC IIKR^KI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKR^KI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKR^KI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKR^KI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKR^KI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity. Key Points MHC II ubiquitination impacts dendritic cell numbers and phenotype. MHC II ubiquitination is critical for Ag-dependent T cell responses in vivo. MHC II ubiquitination is required for Ab responses.