Ena Ladi

Thymic microenvironments for T cell differentiation and selection

The adult thymus provides a variety of specialized microenvironments that support and direct T cell differentiation and selection. In this review, we summarize recent advances in the understanding of the function of microenvironments in shaping a diverse T cell repertoire. In particular, we focus on how thymocytes move in and out of these specialized thymic compartments in response to homing signals, differential chemokine gradients and other factors that regulate T cell migration. In addition, we discuss the diverse developmental signals provided by these microenvironments that contribute to the generation of divergent T cell lineages.

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE+) cells is poorly understood. A model of the differentiation of IgE+ B cells proposes that IgE+ cells develop through a germinal-center IgG1+ intermediate and that IgE memory resides in the compartment of IgG1+ memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE+ cells developed through a germinal-center IgE+ intermediate to form IgE+ memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.

Automated 5-D Analysis of Cell Migration and Interaction in the Thymic Cortex from Time-Lapse Sequences of 3-D Multi-channel Multi-photon Images

This paper presents automated methods to quantify dynamic phenomena such as cell-cell interactions and cell migration patterns from time-lapse series of multi-channel three-dimensional image stacks of living specimens. Various 5-dimensional (x, y, z, t, lambda) images containing dendritic cells (DC), and T-cells or thymocytes in the developing mouse thymic cortex and lymph node were acquired by two-photon laser scanning microscopy (TPLSM). The cells were delineated automatically using a mean-shift clustering algorithm. This enables morphological measurements to be computed. A robust multiple-hypothesis tracking algorithm was used to track thymocytes (the DC were stationary). The tracking data enable dynamic measurements to be computed, including migratory patterns of thymocytes, and duration of thymocyte-DC contacts. Software was developed for efficient inspection, corrective editing, and validation of the automated analysis results. Our software-generated results agreed with manually generated measurements to within 8%.

Thymocyte-Dendritic Cell Interactions near Sources of CCR7 Ligands in the Thymic Cortex

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals.

CCR7 Expression in Developing Thymocytes Is Linked to the CD4 versus CD8 Lineage Decision

During thymic development, T cell progenitors undergo positive selection based on the ability of their T cell Ag receptors (TCR) to bind MHC ligands on thymic epithelial cells. Positive selection determines T cell fate, in that thymocytes whose TCR bind MHC class I (MHC-I) develop as CD8-lineage T cells, whereas those that bind MHC class II (MHC-II) develop as CD4 T cells. Positive selection also induces migration from the cortex to the medulla driven by the chemokine receptor CCR7. In this study, we show that CCR7 is up-regulated in a larger proportion of CD4+CD8+ thymocytes undergoing positive selection on MHC-I compared with MHC-II. Mice bearing a mutation of Th-POK, a key CD4/CD8-lineage regulator, display increased expression of CCR7 among MHC-II-specific CD4+CD8+ thymocytes. In addition, overexpression of CCR7 results in increased development of CD8 T cells bearing MHC-II-specific TCR. These findings suggest that the timing of CCR7 expression relative to coreceptor down-regulation is regulated by lineage commitment signals.

Addendum: IgE + memory B cells and plasma cells generated through a germinal-center pathway

Addendum: IgE + memory B cells and plasma cells generated through a germinal-center pathway

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37 degrees C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

Automatic summarization of changes in image sequences using algorithmic information theory

An algorithmic information theoretic method is presented for object-level summarization of meaningful changes in image sequences. Object extraction and tracking data are represented as an attributed tracking graph (ATG), whose connected subgraphs are compared using an adaptive information distance measure, aided by a closed-form multi-dimensional quantization. The summary is the clustering result and feature subset that maximize the gap statistic. The notion of meaningful summarization is captured by using the gap statistic to estimate the randomness deficiency from algorithmic statistics. When applied to movies of cultured neural progenitor cells, it correctly distinguished neurons from progenitors without requiring the use of a fixative stain. When analyzing intra-cellular molecular transport in cultured neurons undergoing axon specification, it automatically confirmed the role of kinesins in axon specification. Finally, it was able to differentiate wild type from genetically modified thymocyte cells.

5-D imaging and parallel automated analysis of cellular events in living immune tissue microenvironments

The mammalian immune system consists of vital tissue microenvironments that exhibit remarkable structural complexity and dynamic cellular behaviors. It is now possible to acquire time-lapse series of multi-channel three dimensional images of multiple cell types and vasculature simultaneously, revealing dynamic events in their living tissue context. This talk will describe automated image analysis algorithms, parallel computation methods, and large-scale edit-based validation methods to detect and quantify key events such as homogeneous and heterogeneous cell-cell interactions, and methods to map events to their tissue context.