Mechanisms of immune selection of GPI-deficient hematopoietic stem cells in paroxysmal nocturnal hemoglobinuria (PNH)

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by an acquired
mutation of the X-linked PIG-A gene in hematopoietic stem cells, resulting in a
clone of blood cells bearing this mutation. The PIG-A gene is essential for the
expression of glycosyl phosphatidyl inositol (GPI) anchored proteins at the
cell membrane. GPI anchored proteins include complement inhibitors CD55 and
CD59. Deficiency of these molecules on the cell surface of erythrocytes leads
to hemolysis upon complement activation, and associated symptoms such as severe
anemia, hemoglobinuria, abdominal pain, dysphagia and erectile dysfunction.
Next to intravascular hemolysis, patients have a high risk of thrombosis and
many patients have associated bone marrow failure. Symptoms only occur when the
PNH clone has expanded to a certain level. It is not known why a PNH clone
expands.
Interestingly, PNH is closely related to aplastic anemia (AA), which is
considered to result from auto-immune mediated bone marrow damage. A
significant proportion of AA patients have subclinical PNH clones and overt PNH
often develops during the course of AA. Therefore, auto-immune mediated bone
marrow failure may be involved in the pathogenesis of PNH as well. In a setting
of autoimmune mediated bone marrow failure, it is hypothesized that
hematopoietic stem cells deficient in expression of GPI-anchored proteins
survive immunological attack whereas their GPI-positive counterparts do not.
This immunological selective advantage of GPI deficient hematopoietic stem
cells could explain expansion of a PNH clone.

To study the role of GPI-deficiency in susceptibility of hematopoietic stem
cells to cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell mediated
lysis in the pathogenesis of PNH.

We will study differential susceptibility of GPI-positive and GPI-negative
cells for lysis by high-affinity cytotoxic T lymphocytes recognizing antigens
on HSC in vitro. This will be investigated in cytotoxicity assays using cell
lines and hematopoietic stem cells from PNH and AA patients as target cells.
To elucidate the role of individual GPI-linked proteins in causing differential
susceptibility, we will perform blocking studies of GPI-anchored proteins in
cytotoxicity assays, again using cell lines and hematopoietic stem cells from
PNH and AA patients as target cells.
We will characterize potentially autoreactive T cell, Natural Killer (NK) cell
and Natural Killer T (NKT) cell populations in blood samples drawn from PNH
patients using immunophenotyping. Autoreactive T, NK or NKT cell populations
will be expanded and assessed functionally for proliferative capacity, cytokine
production and most importantly, for cytolytic activity towards GPI positive
and GPI negative hematopoietic stem cells.

The risks of a sternal bone marrow aspiration include bleeding and infection
but occur very rarely. Risks of blood drawing include a hematoma, infection or
bleeding at the puncture site.