Faculty Bibliography

Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8(+) and CD4(+) T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8(+) and CD4(+) T cells. Unexpectedly, therapeutic blockade of PD-1-PD-L1 restored CD8(+) T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8(+) T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-gamma. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8(+) T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.

After infection, many factors coordinate the population expansion and differentiation of CD8(+) effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8(+) T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8(+) effector cells. Long-lived memory CD8(+) cells ultimately expressed a small subset of genes shared by natural killer T and gammadelta T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8(+) effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8(+) T cell immunity.

Radiotherapy sensitizes unresponsive tumors to the antineoplastic activity of antibodies that target the inhibitory receptor CTLA-4 on T cells. One molecular mechanism accounting for this therapeutic synergy is the induction of NKG2D ligands on irradiated tumor cells. The fact that NKG2D receptors must be engaged for the elicitation of CD8+ T-cell antitumor responses has important clinical implications.

PROBLEM: Pregnancy is a challenge to the maternal immune system as it allows the growing of a semiallogeneic fetus within the uterus. Such tolerance suggests a set of complex cellular distributions and interactions inside the organ. Until now, direct observation of such processes was absent because proper intravital imaging techniques were not available. METHOD: We developed a new two-photon microscope stage together with a set of surgical procedures to provide direct observation of immune cell within the mouse uterus. RESULTS: Using our technique, we observed an accumulation of dendritic cells (DCs) in the uterus during the estrus phase of the estrus cycle. Some of the observed DC clusters were located near the lumen of the uterus or small blood vessels, each situated on the antimesometrium side. CONCLUSION: While two-photon microscopy has become a widely used technology for intravital imaging, new advances in the development of staging and experimental protocols can still push the limits of this technique for exploring new biology. As proof of this, we demonstrated that with specially designed staging and surgical protocols, we observed the formation of DC clusters in the uterus; structures that may play a role in the complex immunology of the uterus-fetal interface.

Go to www.immunologicalreviews.com to watch an interview with Guest Editor Facundo Batista.

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.

It is increasingly recognized that cell signaling, as a chemical process, must be considered at the local, micrometer scale. Micro- and nanofabrication techniques provide access to these dimensions, with the potential to capture and manipulate the spatial complexity of intracellular signaling in experimental models. This review focuses on recent advances in adapting surface engineering for use with biomolecular systems that interface with cell signaling, particularly with respect to surfaces that interact with multiple receptor systems on individual cells. The utility of this conceptual and experimental approach is demonstrated in the context of epithelial cells and T lymphocytes, two systems whose ability to perform their physiological function is dramatically impacted by the convergence and balance of multiple signaling pathways.

Activation of the Nlrc4 inflammasome results in the secretion of IL-1beta and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8(+) T cell responses than wt L. monocytogenes. It is also known that IL-1beta orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24-48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8(+) T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8alpha(+) DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8alpha(+) DCs, which are known to be critical for T cell response to L. monocytogenes.