Download Characterizing Individual Tissue-Infiltrating T Cell

 2014 Research Grant Program Winning Abstract
Characterizing Individual Tissue-Infiltrating T Cell Clones in the Setting of
Autoimmunity
By Michiko Shimoda
BACKGROUND
Autoimmunity affects nearly 25 million people in the United State and most immune-mediated
diseases remain incurable despite recent advancements in treatment options. This proposal is
based upon the belief that a detailed characterization of the specific T cells driving an
autoreactive inflammatory process will lead to a better understanding of the pathophysiology of
autoimmunity and potentially to the identification of novel targets for future drug development.
Herein, we will take advantage of a novel application that, for the first time, will allow for a highly
focused dissection of the pathogenic T cell repertoire. To date, most studies have focused on
characterizing bulk lymphocyte populations or in vitro cultured T cell clones isolated from
patients with autoimmunity. Unfortunately, flow cytometry studies of peripheral or tissue-derived
lymphocytes are inefficient given that the pathogenic population will comprise only a small
fraction of the analyzed cells. In addition, we have observed that in patients with autoimmunity
and healthy controls there are often many peripherally expanded T cell clones, the significance
of which is unknown. In patients with autoimmunity most of the peripherally expanded T cell
clones do not enter into the site(s) of disease activity. Determining how these expanded but
non-autoreactive T cells differ from the tissue-infiltrating T cells is of considerable importance.
The specific hypothesis being tested is that individual tissue-infiltrating T cell clones can be
characterized by combining cell-sorting strategies with T cell repertoire analysis. This
hypothesis is based upon the following observations. First, in an animal model of autoimmunity,
only a small fraction of the self-directed repertoire actually had the ability to adoptively transfer
disease to naive recipients. Second, this same population could be detected by comparing the
peripheral T cell repertoire to the tissue-infiltrating repertoire. Third, by conducting multiple
magnetic sorts followed by T cell repertoire analysis, the surface phenotype of these cells could
be identified. Based on these observations, the experimental focus of this proposal is to identify
tissue-infiltrating T cell expansions in patients with autoimmunity and then to compare how they
differ from peripherally expanded but non-infiltrative T cells isolated from the same individual.
METHODS
Identification of T cell clonal expansions of interest. As part of an ongoing IRB-approved study,
we are identifying clonotypic T cell expansions in patients with autoimmunity and healthy
controls. Thus far more than 200 patients and controls have been enrolled in this study. When
possible, peripheral T cell expansions are compared to expansions found at a particular site of
pathology, in the case of patients with immune-mediated diseases involving the skin this would
be a biopsy from lesional skin. T cell expansions are then identified using a TCR-specific PCR
designed to amplify all rearranged TCR genes from within a bulk lymphocyte population. Next
generation sequencing libraries are then generated and analyzed using a MiSeq Illumina
sequencer. The current project will build upon this foundation of existing patient-relevant data.
Characterization of individual tissue-infiltrating T cell clones. Tissue-infiltrating T cells can be
characterized from the peripheral blood or inflamed tissue using the following BD-biosciences
product-based approach, which utilizes both BD consumables and BD equipment (BD
FACSAria III and BD LSRFortessa). After T cell expansions have been identified, the next step
will be to obtain some rudimentary knowledge about their cell surface. Using a magnetic bead
separation strategy, the bulk population of lymphocytes will be enrich for cells bearing a
particular activation marker (HLA-DR, CD45RO) or for cells that have lost a lymph node-homing
signal (CD62L, CCR7). T cell repertoire analysis is then repeated on the sorted populations. If
a T cell expansion expresses or lacks a particular surface marker, its frequency will be
increased (or decreased) in the corresponding sorted population. Sorting for the presence or
absence of CD4 or CD8 will also be conducted to determine if the tissue-infiltrating expansions
are of the CD4 or CD8 lineage. Presorting before T cell repertoire analysis allows us to obtain
information about the expanded T cell’s surface, which will be necessary to devise a strategy to
enrich for a particular clone of interest. For example, if we find that a particular T cell expansion
lacks CCR7 and expresses a Vb7-encoded TCR and CD8, we can presort to exclude CD4+ and
CCR7+ cells. The highly enriched population can then be stained with a fluorescently labeled
antibody specific to Vb7 in addition to a panel of fluorescently labeled BD antibodies specific to
multiple other molecules of interest. The labeled cells can then be analyzed by flow cytometry
(BD LSRFortessa). Following this protocol, the resulting cells residing within the Vb7+
population will be virtually clonal, a pure population of a single clone that is known to actively
infiltrate lesional tissue. Using flow cytometry to compare the proteins expressed by these cells
to those expressed by expanded but non-tissue-infiltrating cells, one can gain insight into the
pathophysiology of autoimmunity. Specifically, we will identify surface and intracellular markers
uniquely expressed by the tissue-infiltrating putatively pathogenic clones.
SIGNIFICANCE
Given that a large number of patients have enrolled in this study we are poised to gain insight
into a variety of autoimmune diseases, including scleroderma, psoriasis, and lupus.