Summary While 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 (TPSLM) to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We find that neutrophils form both small, transient or large, persistent swarms via a strikingly coordinated migration pattern. We provide evidence that cooperative action of neutrophils and parasite egress from host cells can trigger swarm formation. Neutrophil swarm formation coincides 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.

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Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laserscanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.neutrophil motility | dynamic imaging | gut | mucosal immunology T oxoplasma gondii infects around a third of humans worldwide and is widely dispersed in other warm-blooded hosts. Although clinical manifestations in the brain, eye, and developing fetus receive the most attention, T. gondii is an oral pathogen and first enters the body and establishes infection in the small intestine. Infection follows consumption of cyst-containing meat or oocyst-contaminated water and produce and is associated with the development of small intestinal pathology in a variety of nonhuman hosts (1). Most notably, experimental infection of C57BL/6 mice by the oral route results in an inflammation of the small intestine that shares immunological features with inflammatory bowel disease (2). This model is useful to further our understanding of host-pathogen interactions in the intestine and of common mechanisms underpinning the development of inflammatory bowel disease (3). Nevertheless, we have limited understanding of how and in which cells infection is established in the intestine, the extent to which the parasite replicates and spreads within the intestine, and how these factors contribute to the development of pathology (2, 4-9). The ability to label living parasites fluorescently and track them in the tissues of infected hosts provides an important tool for investigating these questions (10)(11)(12)(13)(14).Starting in the small intestine, T. gondii must travel long distances and surmount a variety of biological barriers to establish chronic infection in the brain. These barriers include the mucus, the intestinal epithelium, and the blood-brain barrier (7,15). Cells of the immune system are often highly motile and represent attractive transport vessels for pathogens seeking to reach and enter tissues while being protected from the external environment. Consequently, recent studies have focused on the role of immune cells in transporting parasites between tissues (4, 16-23). For example, cluster of differentiation 11b-positive (CD11b + ) cells have been implicated in the dissemination of T. gondii through the blood and across the blood-brain barrier (4, 19). Following oral infection, i...

Summary Highly functional CD8+ effector T (Teff) cells can persist in large numbers during controlled persistent infections, as exemplified by rare HIV-infected individuals who control the virus. Here we examined the cellular mechanisms that maintain ongoing T effector responses using a mouse model for persistent Toxoplasma gondii infection. In mice expressing the protective MHC-I molecule, Ld, a dominant T effector response against a single parasite antigen was maintained without a contraction phase, correlating with ongoing presentation of the dominant antigen. Large numbers of short-lived Teff cells were continuously produced via a proliferative, antigen-dependent intermediate (Tint) population with a memory-effector hybrid phenotype. During an acute, resolved infection, decreasing antigen load correlated with a sharp drop in the Tint cell population, and subsequent loss of the ongoing effector response. Vaccination approaches aimed at the development of Tint populations may prove effective against pathogens that lead to chronic infection.

Thymic regulatory T (Treg) cell production requires interleukin 2 (IL-2) and agonist TCR ligands, and is controlled by competition for a limited developmental niche, but the thymic sources of IL-2 and the factors that limit access to the niche are poorly understood. Here we show that IL-2 produced by antigen-bearing dendritic cells plays a key role in Treg cell development, and that existing Treg cells limit new Treg cell development by competing for IL-2. . Our data suggest that antigen-presenting cells that can provide both IL-2 and a TCR ligand comprise the thymic niche, and that competition by existing Treg cells for a limited supply of IL-2 provides negative feedback for new Treg cell production.

ABSTRACTThe apicomplexan parasiteToxoplasma gondiican cause severe disease in immunocompromised individuals. Previous studies in mice have focused largely on CD8+T cells, and the role of CD4 T cells is relatively unexplored. Here, we show that immunization of the C57BL/6 strain of mice, in which the immunodominant CD8 T cell response to the parasite dense-granule protein GRA6 cannot be generated, leads to a prominent CD4 T cell response. To identify the CD4 T cell-stimulating antigens, we generated aT. gondii-specific,lacZ-inducible, CD4 T cell hybridoma and used it as a probe to screen aT. gondiicDNA library. We isolated a cDNA encoding a protein of unknown function that we call CD4Ag28m and identified the minimal peptide, AS15, which was presented by major histocompatibility complex (MHC) class II molecules to the CD4 T cells. Immunization of mice with the AS15 peptide provided significant protection against subsequent parasite challenge, resulting in a lower parasite burden in the brain. Our findings identify the first CD4 T cell-stimulating peptide that can confer protection against toxoplasmosis and provide an important tool for the study of CD4 T cell responses and the design of effective vaccines against the parasite.

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 To what extent the cortex itself is divided into distinct functional compartments is not yet clear. At one end of the spectrum, it is possible that the cortex is functionally homogeneous. According to this view, cortical thymocytes would invariably be in contact with thymic epithelial cells capable of mediating positive selection, and positive selection could take place with equal probability throughout the cortex. On the other hand, there are indications that cortical thymic epithelial cells are heterogeneous (reviewed in Ref. 7), raising the possibility of functionally distinct regions of the cortex specialized for mediating selection events. The anatomical segregation of thymocytes in the cortex based on their TCR specificity provides an indication for such heterogeneity (8). However, distinct functional regions of the thymic cortex have not yet been identified. Two-photon laser scanning microscopy (TPLSM) provides an opportunity to examine the cellular migration and interaction events in tissues that underlie selection events within the thymus (reviewed in Ref. 9). Initial studies have revealed that thymocytes undergo a calcium-dependent stopping in response to positive selection signals in the thymus (10) and that interaction of thymocytes with MHC-bearing stromal cells can lead to prolonged but dynamic cell-cell contacts (11). Additionally, analysis of cell migration in the cortex of intact thymic lobes provided evidence that thymocytes move via a random walk in the cortex before positive selection and undergo rapid, directed migration to the medulla as a result of positive selection (12). These studies provide an early glimpse into the regulation of thymocyte migration by positive selection. However, the interactions of thymocytes with other cell types in the intact thymus and how these interactions change during the process of positive selection have not yet been addressed.Herein we examine the impact of the chemokine receptor CCR7 and TCR repertoire selection on thymocyte-DC interactions in the cortex of intact thymic lobes. We show that thymic DCs form intimate associations with cortical capillaries near sources of CCR7 ligands in the cortex. We find that thymocytes extensively

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.