Tracking Alloreactive B-cell Responses Público

Karpenshif, Yoav (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/8336h2602?locale=es
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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

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