Ellen A. Robey

An Activated Form of Notch Influences the Choice between CD4 and CD8 T Cell Lineages

Notch is a transmembrane receptor that controls cell fate decisions in Drosophila and whose role in mammalian cell fate decisions is beginning to be explored. We are investigating the role of Notch in a well-studied mammalian cell fate decision: the choice between the CD8 and CD4 T cell lineages. Here we report that expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision.

Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes.

The cellular dynamics underlying activation of CD8+ T cells by dendritic cells (DCs) in the lymph node are not known. Here we have tracked the behavior of T cells and DCs by subjecting intact lymph nodes to real-time two-photon microscopy. We show that DCs scan at least 500 different T cells per hour in the absence of antigen. Antigen-bearing DCs are highly efficient in recruiting peptide-specific T cells and can engage more than ten T cells simultaneously. The duration of these interactions is of the order of hours, not minutes. The overall avidity of the interaction influences the probability that T cells will be stably captured by DCs, providing a possible basis for T cell competition. Taken together, our results identify the cellular behaviors that promote an efficient CD8+ T cell response in the lymph node.

Notch Activity Influences the αβ versus γδ T Cell Lineage Decision

Abstract The choice between the αβ or γδ T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as γδ T cells than as αβ T cells, implying that reduced Notch activity favors the γδ T cell fate over the αβ T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional γδTCR gene rearrangements to adopt the αβ T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the αβ versus γδ T cell lineage decision.

Dynamics of neutrophil migration in lymph nodes during infection.

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

A decade of imaging cellular motility and interaction dynamics in the immune system.

The Immune System in Three Dimensions Immune cells must traffic within the tissues in which they reside and also through the bloodstream and lymphatic system in order to defend the host against infection. Until 10 years ago, immunologists had very little idea about how the immune response was coordinated in three dimensions. This all changed with the application of two-photon microscopy, applied intravitally or on tissue explants, to the immune system. Germain et al. (p. 1676) review how studies using this technology have informed our knowledge of immune system dynamics and discuss how to apply this technology in the future to gather further insights. To mount an immune response, lymphocytes must recirculate between the blood and lymph nodes, recognize antigens upon contact with specialized presenting cells, proliferate to expand a small number of clonally relevant lymphocytes, differentiate to antibody-producing plasma cells or effector T cells, exit from lymph nodes, migrate to tissues, and engage in host-protective activities. All of these processes involve motility and cellular interactions—events that were hidden from view until recently. Introduced to immunology by three papers in this journal in 2002, in vivo live-cell imaging studies are revealing the behavior of cells mediating adaptive and innate immunity in diverse tissue environments, providing quantitative measurement of cellular motility, interactions, and response dynamics. Here, we review themes emerging from such studies and speculate on the future of immunoimaging.

Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum

The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2Ld major histocompatibility complex class I molecule. Presentation of the HF10–H-2Ld ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8+ T cell populations was impaired in T. gondii–infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.

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.

Notch in vertebrates

Homologs of the Notch receptor and its ligands participate in cell fate decisions during vertebrate development. The past year has seen significant advances in knowledge of the role of Notch in Xenopus neuronal development and T-cell development and in our understanding of the Notch signalling pathway in vertebrates. Connections have also been discovered between alterations in Notch function and human disease.

Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node.

Memory T cells circulate through lymph nodes where they are poised to respond rapidly upon re-exposure to a pathogen; however, the dynamics of memory T cell, antigen-presenting cell, and pathogen interactions during recall responses are largely unknown. We used a mouse model of infection with the intracellular protozoan parasite, Toxoplasma gondii, in conjunction with two-photon microscopy, to address this question. After challenge, memory T cells migrated more rapidly than naive T cells, relocalized toward the subcapsular sinus (SCS) near invaded macrophages, and engaged in prolonged interactions with infected cells. Parasite invasion of T cells occurred by direct transfer of the parasite from the target cell into the T cell and corresponded to an antigen-specific increase in the rate of T cell invasion. Our results provide insight into cellular interactions during recall responses and suggest a mechanism of pathogen subversion of the immune response.

A Reassessment of the Effect of Activated Notch1 on CD4 and CD8 T Cell Development

The Notch signaling pathway plays an important role in the early steps of T cell development and in the generation of T cell tumors, but its role in the CD4 vs CD8 lineage decision is controversial. Notch1 is not essential for CD4 or CD8 T cell development; however, there are suggestions that multiple Notch family members may act in a redundant fashion during thymic development. In theory, expressing a constitutively activated form of Notch in CD4+CD8+ thymocytes could provide clues about the normal role of Notch in developing CD4 and CD8 T cells. Unfortunately, two different studies of transgenic mice expressing activated forms of Notch1 (Notch1IC) led to conflicting conclusions. In this study, we re-examine the effect of the two Notch1IC transgenes on thymocyte development. We find that both Notch1IC transgenic lines display a decrease in CD4 single positive (SP) thymocytes and a corresponding increase in CD8 SP thymocytes. The enhanced development of CD8 SP thymocytes is dependent on either class I or II MHC. Thus, data from two different Notch1IC transgenic lines indicate that Notch activity promotes CD8 and inhibits CD4 SP development. We suggest that the discrepancies in previous reports of Notch1IC transgenic mice are due to differences in the propensity of the two different transgenic lines to develop tumors.