Abstract 2235


Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease characterized by hemolysis due to an acquired mutation in the X-linked PIG-A gene in the hematopoietic stem cell (HSC). This leads to a clone of hematopoietic cells with deficient expression of glycosyl phosphatidyl inositol (GPI) anchored proteins at the cell membrane. The clinical evolution of PNH arises through clonal expansion of PIG-A mutated HSC which is insufficiently explained by the PIG-A mutation alone. Hypothetically, clonal expansion could result from autoreactive T cells selectively attacking normal HSC, whereas GPI deficient HSC are unharmed.

Methods and Results:

we investigated the presence of potentially autoreactive T cells in peripheral blood of patients with PNH (n = 39) by flow cytometry. We compared T cell subset frequencies and absolute numbers with healthy controls (n = 25) using Mann-Whitney U test. In PNH patients, T cells expressing the NK cell marker CD56 were significantly elevated, both in percentage (p < 0.001) and in absolute numbers (p < 0.01). Furthermore, the frequency of T cells expressing the activating NK cell receptors (NKRs) NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01) was significantly increased. KIR2DS4+, NKG2C+ and NKG2D+ T cells mainly consist of highly differentiated effector memory CD45RA+ T cells (TEMRA) (KIR2DS4: median 90%, range 70–96%, NKG2C: median 83%, range 49–99%, NKG2D: median 40%, range 38–66%). A highly variable proportion of these T cell populations consists of γδ T cells (KIR2DS4: median 28%, range 6–72%, NKG2C: median 36%, range 3–75%, NKG2D: median 11%, range 7–40%). By 10 color flow cytometry, we examined NKR coexpression patterns. KIR2DS4+ and NKG2C+ T cells mainly coexpress either only NKG2D (KIR2DS4+ T cells: median 24%, range 2 – 74%, NKG2C+ T cells: median 21%, range 3–71%), or a combination of NKG2D, NKG2C and CD158b1/b2,j, but not inhibitory NKG2A (KIR2DS4+ T cells: median 16%, range 1 – 55%, NKG2C+ T cells: median 20%, range 1–60%). In contrast, NKG2D+ T cells generally do not express any other NKRs tested (median 77%, range 53–84%). NKG2D+ KIR2DS4+ cytotoxic T lymphocyte (CTL) lines isolated from PNH patient peripheral blood and bone marrow display high cytolytic activity towards CD34+ hematopoietic progenitor cell lines and MHC class I deficient K562 cells, suggesting T cell receptor independent cytolytic activity. These NKR+ CTL lines are capable of differentially lysing GPI+ and GPI- hematopoietic cell lines, however not in all cell line models and CTL lines. This suggests that multiple factors, as for example the highly activated status of in vitro cultured CTLs, influence whether or not GPI dependent lysis occurs.


The increased frequency of T cells expressing activating NK cell receptors KIR2DS4, NKG2C, and NKG2D, with a CD8+ effector-memory phenotype and differences in cytotoxicity towards GPI+ and GPI- hematopoietic cell lines suggests that these T cell populations may be involved in bone marrow failure and expansion of PNH clones.


Muus: Alexion: member of advisory board.

Author notes


Asterisk with author names denotes non-ASH members.