Faculty Bibliography

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

Cells communicate by sensing diffusible or surface-associated chemical signals, with the surface-associated molecules also providing mechanical cues. New work now shows that both chemical and mechanical signals are critical for the communication between T cells and antigen-presenting dendritic cells in the initiation and maintenance of immune responses.

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCgamma1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

Integrative analytical approaches are needed to study and understand T cell motility as it is a highly coordinated and complex process. Several computational algorithms and tools are available to track motile cells in time-lapse microscopy images. In contrast, there has only been limited effort towards the development of tools that take advantage of multi-channel microscopy data and facilitate integrative analysis of cell-motility. We have implemented algorithms for detecting, tracking, and analyzing cell motility from multi-channel time-lapse microscopy data. We have integrated these into a MATLAB-based toolset we call TIAM (Tool for Integrative Analysis of Motility). The cells are detected by a hybrid approach involving edge detection and Hough transforms from transmitted light images. Cells are tracked using a modified nearest-neighbor association followed by an optimization routine to join shorter segments. Cell positions are used to perform local segmentation for extracting features from transmitted light, reflection and fluorescence channels and associating them with cells and cell-tracks to facilitate integrative analysis. We found that TIAM accurately captures the motility behavior of T cells and performed better than DYNAMIK, Icy, Imaris, and Volocity in detecting and tracking motile T cells. Extraction of cell-associated features from reflection and fluorescence channels was also accurate with less than 10% median error in measurements. Finally, we obtained novel insights into T cell motility that were critically dependent on the unique capabilities of TIAM. We found that 1) the CD45RO subset of human CD8 T cells moved faster and exhibited an increased propensity to attach to the substratum during CCL21-driven chemokinesis when compared to the CD45RA subset; and 2) attachment area and arrest coefficient during antigen-induced motility of the CD45A subset is correlated with surface density of integrin LFA1 at the contact.

There is significant evidence that brain-infiltrating CD8+ T cells play a central role in the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the mechanisms through which they mediate their pathogenic activity during malaria infection remain poorly understood. Utilizing intravital two-photon microscopy combined with detailed ex vivo flow cytometric analysis, we show that brain-infiltrating T cells accumulate within the perivascular spaces of brains of mice infected with both ECM-inducing (P. berghei ANKA) and non-inducing (P. berghei NK65) infections. However, perivascular T cells displayed an arrested behavior specifically during P. berghei ANKA infection, despite the brain-accumulating CD8+ T cells exhibiting comparable activation phenotypes during both infections. We observed T cells forming long-term cognate interactions with CX3CR1-bearing antigen presenting cells within the brains during P. berghei ANKA infection, but abrogation of this interaction by targeted depletion of the APC cells failed to prevent ECM development. Pathogenic CD8+ T cells were found to colocalize with rare apoptotic cells expressing CD31, a marker of endothelial cells, within the brain during ECM. However, cellular apoptosis was a rare event and did not result in loss of cerebral vasculature or correspond with the extensive disruption to its integrity observed during ECM. In summary, our data show that the arrest of T cells in the perivascular compartments of the brain is a unique signature of ECM-inducing malaria infection and implies an important role for this event in the development of the ECM-syndrome.