Using intravital 2-photon imaging methods developed in the LBS, we can routinely image peripheral organs and tissues so that immune effector cell behavior during infectious processes can be observed. Using these new methods, we previously described lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver and showed that only a small fraction of the antigen-specific T cells within a granuloma undergo migration arrest at any time. This small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma. These findings provided entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. Many of these observations have been repeated in M tuberculosis-infected animals. The finding that only a small fraction of the cells being imaged in a tissue are actually engaged in effector function at any time and that these have a dynamic behavior distinct from the bulk of the imaged cells raises critical questions about many existing and ongoing studies in other laboratories using intravital 2-photon imaging, in which the bulk or average behavior of clonally-related cells is taken as representative of the functional population. We have published studies of T-cell motility in infected lungs, comparing the response of CD4+ T cells to influenza vs. BCG infection and addressing the question of whether the same limited activation of effector function is seen as in the liver and whether this varies with the pathogen. These studies confirmed the conclusions of our liver granuloma model, in that only a very small fraction of the mycobacteria-specific T cells arrest movement and produce cytokine (IFN-gamma) in this infected tissue setting. In striking contrast, the influenza antigen-specific T cells showed much greater fractional migration arrest and a correspondingly greater fraction of cytokine producing T-cells. These latter results emphasize that the extent of the effector response varies greatly depending on the microbe, in apparent concordance with the density of presented antigen (influenza >> mycobacteria). In other studies, we are examining how the structure of lymph nodes is organized to limit access of invading organisms to the blood stream. These studies showed a crucial role of CD169+ subcapsular sinus macrophages in responding to lymph borne pathogens by producing the cytokine IL-18, which together with others such as IL-12 or type 1 interferon, resulted in rapid cytokine-induced cytokine production by a diverse set of lymphocytes (memory CD8 T cells, gamma-delta T cells, NK cells, and NKT cells). IFNgamma plays a critical role in preventing replication of incoming bacteria in the lymph node and in preventing systemic dissemination. Remarkably, these various lymphocytes population, while highly motile, show constrained localization to the regions near to the subcapsular sinus-lining CD169+ macrophages, revealing a very specific tissue micro-anatomy that supports robust innate responses to incoming pathogens to present their dissemination. More recently we have begun exploration of the interaction of microbes with the host gut, in particular, examining how potential pathobionts are constrained to exist in a commensal-like homeostatic state with the host through the action of a combination on innate lymphocytes (ILC) and T cells (see also ZIA AI000545-32 LSB). Combining our new static imaging tools with the use of various reporter and genetic engineered strains of mice, we have been able to quantify the extent of cytokine signaling of epithelial cells in the small bowel resulting from IL-23 activation of IL-22 production from ILC3 based on pSTAT3 presence and to determine that these ILC operate in conjunction with Th17 and Treg to shape the normal commensal microbiota. We have now shown that ILC3s and adaptive CD4 T cells participate sequentially to establish the mature state of non-inflammatory commensalism with certain bacteria. Strikingly, the ILC3 and CD4+ T cells use different mechanisms in regulating bacterial numbers, as evidenced by a lack in wild-type mice of the extensive pSTAT3 signature seen in RAG mice. This raises doubts about the usual assumption that ILC subsets and CD4 effector subsets share effector mechanisms but differ only in whether an antigen-specific receptor is used to drive activation. Further, we have discovered that one of the reasons evolution employs ILCs early then switches to adaptive T cell immunity over time is to prevent persistent IL-22 production by ILC3s, which causes epithelial cells to decrease lipid transporter expression, resulting in loss of adipose tissue and blood lipid dysregulation in the host. The imaging methods developed for this project have also been employed to discover the site of IL-25 action and to detail the proliferation and migration of activated ILC2 from gut to peripheral organs where they mediate anti-helminth protective immunity. These studies show that in contrast to the usual assumption that ILC are tissue resident effector cells, they have a similar behavior to adaptive T cells in that they are activated, proliferate, and differentiate in one tissue (in the case of iILCs, the small bowel lamina propria), the migrate into lymph and then the blood circulation in an S1P-dependent manner to exit at a distant tissue to mediate protective function. The molecular mechanisms employed by the ILC2s to leave the intestine is thus the same as that employed by adaptive T cells to exit the lymph node after activation, involving S1PR sensing of an S1P gradient between the tissue and blood. The drug FTY720 prevents ILC2s from exiting the intestine after IL-25 activation, preventing them from mediating effective protection against helminth infection involving the lungs, to which the activated ILC2s usually travel. We have examined the cellular basis for strong host protection against malaria infection in a mouse model using a push-pull vaccination scheme developed in the laboratory of Prof. Adrian Hill at University of Oxford. These studies have revealed a key role for CD8 T cells with a tissue resident memory cells phenotype in terms of CD69 expression along with chemokine receptor expression consistent with liver homing. Imaging studies have revealed how these effectors patrol the liver and how they cluster around infected hepatocytes to mediate elimination of the infected cells once detected. As part of ZIA AI000545-32 LISB, we have used this malaria infection model to reveal the critical role an Ebi2-gradient in lymph nodes plays in pre-positioning naive CD4+ T cells for optimal interaction with antigen-presenting cDC2 in the periphery of the node. Without this spatial organization, CD4+ T cell help for CD8+ memory fails and post-vaccination resistance to malaria is lost. Finally, we have returned to the study of lethal influenza infection. Using data from our previous systems-level analysis, we examined whether tissue based RNA expression data could inform a blood signature that would allow prediction of lethal infection at an early stage. While we could validate the specificity of the lethal vs. influenza infection pan signature, we could not reliably predict lethality at one LD50 in infected mice with just innate immune components in the signature. However, addition of markers for activated T cells did permit prediction of which animals would die after an LD50 infection, suggesting that stochastic variation in antigen specific immune repertoire among the infected animals can determine the difference between life and death, with important implications for understanding many lethal human viral infections.
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