Abstract
Modern medicine has become increasingly aware of the detrimental
effects that the B-cell mediated humoral response has on
allografts. Unfortunately, the lack of knowledge about the signals
and mechanisms that govern this response has impeded the
development of an effective clinical treatment to counter it. This
research presents a proof of concept of a murine transplant model
system that is well suited to observe the B-cell mediated humoral
response to donor tissue. The system utilizes a combination of
advanced flow cytometric techniques, ELISA, and ELISPOT assays to
phenotype donor specific B-cells, track the kinetics of the
response, and quantify as well as qualify donor specific
antibodies. The use of the model system has made it possible to map
the kinetics of the humoral response to donor antigen and
demonstrate that a difference in duration of antigen exposure
affects the long-term humoral alloreactivity of an organism.
Furthermore, the model system has allowed for the phenotyping of
donor specific memory B-cells and has allowed for the
identification of an IgM expressing subset of memory B-cell that
has yet to be identified in a murine system. Lastly, the system was
applied to T-cell receptor knock out transgenic mice to demonstrate
that a long-term humoral response to allograft occurs in the
absence of T-cell help.
Table of Contents
Introduction...........................................................................................
1
Methods
................................................................................................
8
Mice.......................................................................................................
8
H-2 Tetramer preparation
..........................................................................
8
Flow
Cytometry........................................................................................
9
Enzyme Linked Immunosorbent
Assay-ELISA..................................................10
Enzyme Linked Immunosorbent Spot-ELISPOT
...............................................12
Results
................................................................................................
14
Novel methods to track donor specific humoral immunity
................................. 14
Defining the
Donor-specific Memory B-Cell Response
.......................................18
Applying the
model system-Tracking the T-cell independent response
............... 22
Discussion
............................................................................................
24
A new model system-Its virtues and proof of concept
.................................... 24
Applying the
model-TCRKO
mice.................................................................
29
Future
research.......................................................................................
31
References:
..........................................................................................
33
About this Honors Thesis
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