Doctoral Thesis: Microfluidic single-cell technologies for assaying lymphocyte interactions

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Event Speaker: 

Burak Dura

Event Location: 

4-270

Event Date/Time: 

Tuesday, July 7, 2015 - 10:30am

Abstract: 

Immune cells do not live in isolation but interact to coordinate and execute
their many functions. One of the chief routes they foster communication is
through direct physical contact that enables them to read and interpret
signals mediated at membrane interfaces. Despite the critical importance of
these direct interactions in determining crucial developmental and
functional immunological responses, their dynamic nature together with vast
heterogeneity and polyfunctionality of individual immune cells have
presented technical challenges for their systematic investigation. In
particular, only limited tools are available that can exert control over the
individual cells and their microenvironments to be able to precisely define
interactions and deeply profile their outcomes at the individual cell level
to resolve emerging immune responses within each single-cell. 
To fill this critical void, this thesis presents the development and
implementation of novel microfluidic technologies for single-cell analysis
of direct cell-cell interactions in immunology. By combining carefully
designed weir-based hydrodynamic traps with a multistep cell loading
procedure, the microfluidic devices capture and controllably pair hundreds
of cells in parallel. This approach provides requisite control over
interactions with one-to-one interacting partners, well-defined and
synchronous initiation of interactions, and enduring contacts. It also
provides full control over the soluble microenvironment by solution exchange
without losing cell registration. Accordingly, these features enable
monitoring and assaying lymphocyte interactions longitudinally from the
beginning with multiparametric single-cell measurements. These capabilities
in turn allow probing into complete immune cell activation window from the
very onset for direct correlation analyses within hundreds of individual
cells in a single experiment. 

We apply these new 'microfluidic cell pairing' technologies to quantitative
investigation of lymphocyte interactions to elucidate lymphocyte activation
dynamics and their relation to diverse functional behaviors at the
single-cell level. These studies help resolve qualitatively and
quantitatively distinct calcium signaling patterns in single CD8 T cells
based on varying T cell receptor affinities which correlate with
differential cytokine output. Similar studies with natural killer (NK) cells
identify a previously unreported inverse correlation between the strength of
early calcium signaling and cytokine production, and further indicate a
calcium-dependent mechanism for selective regulation of cytotoxicity and
cytokine production in NK cells. Collectively, these findings provide
essential insight into the regulation and evolution of immune responses
within individual immune cells, and establish the potential of these new
microfluidic technologies to address important questions on many aspects of
cell-cell interactions across biology in general and in immunology in
particular.
 
Thesis Supervisor: Prof. Joel Voldman
Thesis Committee: Profs. Hidde L. Ploegh, Sangeeta N. Bhatia