Download Unraveling the Tissue Specific Antigen Presentation That Results in

2011 Research Grant Program Winning Abstract
Unraveling the Tissue Specific Antigen Presentation That Results in the
Systemic Induction of Tolerance during Hepatic Gene Therapy
By Brad Hoffman
A large number of studies in experimental animal models have demonstrated the
significant potential of adeno-associated virus (AAV) vectors as a therapeutic tool for in
vivo gene transfer. Unfortunately, there has been only minimal success in translation of
these results into clinical studies. Data obtained from the animal studies and recent
human clinical trials suggests that the adaptive immune responses pose a significant
hurdle in future clinical application. Additionally, it is now believed that the innate
immune response may actually have a more essential role in directing the process than
previously thought. Accordingly, the ability to maintain immunological unresponsiveness
to the therapeutic protein is a key requirement for successful therapy.
The liver has a distinctive composition and microenvironment that favors the
development of immune tolerance above that of immunity. Antigens expressed in situ
are more likely to be tolerogenic than those expressed in the periphery. However, the
mechanism(s) that determine the balance between intrahepatic immunity and tolerance
are poorly understood. Traditionally, activation of T cells occurs in lymphoid tissues such
as the lymph nodes (LNs) or the spleen. Nonetheless, the liver also contains various
cells that are capable of directly presenting antigen within MHC to CD4+ T cells. The
most abundant liver resident APCs are the Kupffer cells (KCs), dendritic cells (DCs), and
liver sinusoidal epithelial cells (LSECs), all of which have been shown to participate in
the induction of immune tolerance. The DCs are considered well equipped to initiate and
regulate immune activity. Unlike traditional splenic DCs, the liver contains several
different subsets of DCs, each of which possesses a unique phenotype and functional
maturation status, which is important to their ability to trigger immune responses.
Insufficiently activated dendritic cells stimulate immune tolerance, whereas complete
activation results in the induction of antigen-specific immunity.
Objectives:
We hypothesize that the induction of immunity or tolerance is determined by a
competition between the liver and secondary lymphoid tissues for the primary activation
of T cells. In other words, the initial/dominant site of antigen presentation governs the
final immune response. Typically, a destructive immune response occurs when the initial
activation of lymphocytes occurs within the LNs. In contrast, it appears that tolerance is
favored if antigen presentation and T-cell activation initially occur in the tolerogenic
microenvironment of the liver. The research being proposed will provide a unique and
innovative assessment of a murine model of hepatic AAV gene transfer. Our objective is
to 1) assess and phenotypically characterize the hepatic microenvironment, and 2)
identify tissues where antigen presentation and early proliferation of T cells occurs and
track their migration following intrahepatic gene transfer. Ultimately, the goal is to
improve the overall understanding of how to induce (or prevent) unresponsiveness to a
transgene product, which is a key requirement for successful gene therapy.
Specific Aim 1:
Our preliminary data suggests that there is a direct correlation between the magnitude of
hepatic transgene expression and the ability to confer antigen-specific peripheral
tolerance to the transgene product. Using BD™ ELISA reagents and BD™ Cytometric
Bead Arrays, we will profile the microenvironment of the liver in mice that have received
various doses of AAV vector. Then lymphocytes/monocytes will be isolated from the
perfused livers, spleens, and LNs of mice that have received either tolerogenic, subtolerogenic, or null doses of AAV vector with the assistance of BD IMag™ cell
enrichment kits. Phenotypic and functional analyses of these cells using panels of
fluorescently conjugated BD antibodies and cytometry reagents will be performed by
multiparametric flow cytometry using a BD™ LSR II flow cytometer with BD FACSDiva™
software:
DC subsets (cDC vs pDC): CD3, CD11c CD11b CD45R/B220 PDCA-1
APC subsets (KC vs LSEC): CD3, F4/80, CD11c, CD11b, Ly-6G/C, CD146
T cells: CD3, CD4, CD8, CD25, CD27, CD127, PD-1, CD62L, CD44, CD69, CD103
Regulatory T cells: CD3+, CD4+, CD25+, FoxP3+, Helios, CD27, CD127
Specific Aim 2:
Identify tissues where early antigen presentation and proliferation of CD4+ T cells occur
during hepatic gene replacement therapy. The goal of this aim is to demonstrate that if
antigen is overwhelmingly presented in the liver by a resident APC, tolerance is the
preferred outcome. Whereas, if early antigen presentation were slow or limited, allowing
DCs to migrate to the LNs, that immunity would prevail and result in hepatocellular
injury. We will take advantage of a TCR transgenic model by using adoptively
transferred CFSE-pulsed T cells to identify the specific tissue location of early in vivo
proliferation. In addition, we will monitor T-cell migration and proliferation at various time
points after hepatic gene transfer. Five days after gene transfer, lymphocytes harvested
from the liver, spleen, draining LNs, and non-draining LNs (as controls) will be assayed
for in vivo T-cell proliferation by flow cytometric analysis (BD LSR II cytometer and BD
FACSDiva software) using PE-conjugated clonotypic KJ1-26 monoclonal antibody (BD
Biosciences) and detection of the CFSE dye.
Summary and Clinical Significance:
Gene therapy studies have identified several possible immunological outcomes. The
experimental results from this BD Immunology Research Grant proposal will increase
our knowledge of the immune-specific mechanisms associated with hepatic gene
transfer and ought to lay the foundation for additional research that will link tissue
specific (hepatic) antigen presentation to the induction of T-cell tolerance.
The BD Biosciences Research Grant Program aims to reward and enable important
research by providing vital funding for scientists pursuing innovative experiments to
advance the scientific understanding of disease.
Visit bdbiosciences.com/grant to learn more and apply online.