M. Anna Zal

T cell receptor binding kinetics and special role of Vα in T cell development and activation

The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important role in determining thymocyte-positive and-negative selection in the thymus, as well as in T cell activation. The α chain of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory and otherson the role of poly-morphism in the Vα combining site in determining MHC class restriction, and on kinetic parameters in thymocyte selection.

Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells.

We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4‐64 (N‐(3‐triethylammoniumpropyl)‐4‐(p‐diethylaminophenylhexatrienyl)‐pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620–650 nm in contrast to 480–520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual‐excitation imaging of FM4‐64 in relation to lamina‐associated polypeptide‐1–green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long‐wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4‐64 as the first vital dye sensitive to the NE environment opens new ways for real‐time visualization and functional studies of the NE.

Interleaved dual-wavelength multiphoton imaging system for heterologous FRET and versatile fluorescent protein excitation

Broad two-photon cross sections of fluorescent proteins allow excitation with a single wavelength of a tunable femtosecond pulsed laser but the brightness is sub-optimal and the cross-talk prevents sensitized emission FRET imaging in heterologous systems. We present a novel arrangement of a resonant scanning microscope capable of fast interline dual wavelength femtosecond excitation of pairs of fluorophores. This allows optimal and selective excitation of mCerulean and mCitrine as well as FRET imaging using the principles of sensitized emission 3-cube imaging. Performance of the system in thin and thick specimens is discussed.

Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes

ABSTRACT Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Epidermal T Cell Dendrites Serve as Conduits for Bidirectional Trafficking of Granular Cargo

Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC’s long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1+ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered.

Abstract 4290: Visualizing immune surveillance in lung metastasis progression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL As stipulated by the Paget and Fuchs’ “seed and soil” analogy, the establishment of metastases depends on the microenvironment of target tissue; as such, it also depends on the tissue immunosurveillance system. Anti-tumor immune responses are subdued in advanced tumors by various immune suppressory mechanisms, such as by tumor associated CD4 FoxP3 regulatory T-reg cells. But how immune suppression sets in at the sites of metastasis, and how it could be overcome there remains unclear. Using multiphoton and confocal microscopy, we have visualized the immune cells dynamics that is triggered by various metastatic cancers in the lungs. We found that single, blood-carried cancer cells that settle in the capillary bed triggered strikingly rapid recruitment of CD11c dendritic cells (DCs), which were heterogeneous. Some of those DCs wrapped around the singular cancer cells while other probed their intracellular content by acquiring tumor-derived vesicles. These phagocytic DCs then carried away their cargo towards the draining lymph nodes, thereby initiating tumor antigen cross-presentation. After 24-48 hours, micrometastases became surrounded by T cells that were highly enriched in T-reg. The motility patterns and cell depletion experiments showed that T-reg largely ignored tumor cells but were attracted by the phagocytic tumor-associated DCs. In result of these interactions, the phagocytic DCs became the foci of highly dynamic three-cell interactions between T-reg and the effector T cells, sequestering most T cells at the tumor margin. In this context, we tested the impact of immunomodulatory therapies. The immune cell dynamics that we uncovered in metastases was responsive to therapeutic immune modulation aimed at the tumor-associated phagocytic DCs and also at Programmed Death (PD-1) pathways. Taken together, our intravital dynamic imaging results highlight the duality of metastasis-associated phagocytic DCs. On the one hand, these cells provide tumor antigen cross-presentation and recruit T cells to nascent secondary tumors, but on the other hand, they aid metastasis progression by promoting early on the contact of tumor-infiltrating T cells with T-reg. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4290. doi:1538-7445.AM2012-4290