Jenny O. Ross

Distinct Temporal Patterns of T Cell Receptor Signaling During Positive Versus Negative Selection in Situ

Thymocyte survival and maturation in the thymus depend on transient signaling and thymocyte migration. Thymocytes Make a Deadly Stop Thymocytes are T cell precursors that develop in the thymus. To ensure that mature T cells discriminate appropriately between self-antigens and foreign antigens, thymocytes undergo selection initiated by the presentation of self-peptides by antigen-presenting cells to the T cell receptor (TCR) on the thymocyte (see the Perspective by Brzostek and Gascoigne). Relatively weak TCR signaling ensures thymocyte survival (positive selection); however, strong TCR signaling results in thymocyte death (negative selection). Melichar et al. performed two-photon imaging of fluorescently labeled mouse thymocytes on thymic slices containing various antigenic peptides to simultaneously monitor thymocyte migration and the extent of intracellular Ca2+ signaling. Positive selection was characterized by migratory thymocytes with decreased Ca2+ signaling, whereas negative selection was characterized by migratory arrest and enhanced Ca2+ signaling, suggesting that transient TCR signaling without migratory arrest is required for thymocyte survival and development. The recognition by the T cell receptor (TCR) of self-peptides presented by the major histocompatibility complex (MHC) on antigen-presenting cells, such as dendritic cells and thymic epithelial cells, controls T cell fate in the thymus, with weak TCR signals inducing survival (positive selection) and stronger signals inducing death (negative selection). In vitro studies indicate that peptide ligands that induce positive selection stimulate a low, but sustained, pattern of TCR signaling; however, the temporal pattern of TCR signaling in MHC class I–restricted thymocytes (thymocytes that are presented with peptides by MHC class I) in the thymus, under conditions that support positive selection, is unknown. We addressed this question by examining intracellular Ca2+ dynamics and migratory changes in thymocytes undergoing positive and negative selection in thymic slices. Brief, serial signaling events that were separated by migratory periods and low cytosolic Ca2+ concentrations correlated with the positive selection of MHC class I–restricted thymocytes, whereas sustained Ca2+ signaling and the arrest of thymocytes were associated with negative selection. Low-avidity peptides and the presentation of peptides by cortical thymic epithelial cells, rather than dendritic cells, failed to induce strong migratory arrest of thymocytes, which led to transient TCR signaling. Thus, we provide a comparison of positive and negative selection signals in situ and suggest that the absence of strong stop signals distinguishes between positive and negative selection.

Opposing chemokine gradients control human thymocyte migration in situ

The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection.

Two‐Photon Imaging of the Immune System

Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two-photon microscopy that are intended to introduce the new user to some basic aspects of this method.

Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Significance Developing T cells are positively selected in the thymus to ensure that their antigen receptors can interact with self-MHC. For CD8 T cells, this process takes days to complete, yet the steps involved are poorly understood. We followed a synchronized wave of cells undergoing positive selection within three-dimensional thymic tissue. Surprisingly, migration from the cortex to the medulla occurred before CD4 down-regulation and while thymocytes still required TCR signaling for efficient positive selection. There was a gradual change in the pattern of calcium signaling over time, with an upward shift in basal intracellular calcium correlating with increased speed and brief signaling events. Our data have interesting implications for how positive and negative selection shape the mature CD8 T-cell repertoire. Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3–4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca2+]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca2+]i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.

Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo.

Studying T Cell Development in Thymic Slices.

Recently, tissue slices have been adapted to study both mouse and human T cell development. Thymic slices combine and complement the strengths of existing organotypic culture systems to study thymocyte differentiation. Specifically, the thymic slice system allows for high throughput experiments and the ability to introduce homogenous developmental intermediate populations into an environment with a well-established cortex and medulla. These qualities make thymic slices a highly versatile and technically accessible model to study thymocyte development. Here we describe methods to prepare, embed, and slice thymic lobes to study T cell development in situ.

Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection.

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell–cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte–thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection.