Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria

Abstract

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Figure 1

Method of CD34⁺ cell collection and phenotype of CD34⁺ cell progeny from two healthy controls. (a and b) Gates were placed on forward scatter and side scatter (shown by the box in a) and CD34⁺ fluorescence. The purity of the sorted cells is shown in b. (c–f) After culturing, cells were stained for GPI-anchored CD59; CD45; and DNA in three-color analyses. The results for two healthy volunteers, N1 (c and e) and N2 (d and f) are shown.

Figure 2

Flow cytometric analyses of progeny of normal CD34⁺, patients’ PIGA^– CD34⁺, and patients’ PIGA⁺ CD34⁺ cells after 11 days of growth in liquid culture. The cells were stained as in Figure 1. The left (a, c, and e) and right (b, d and f) panels show CD59 vs. CD45 and CD59 vs. DNA, respectively. The upper panels (a and b) show the same patterns for normal cells seen in the preliminary studies. As seen in the middle panels (c and d), the progeny of PIGA⁺ cells were uniformly CD59⁺, and as seen in the lower panels (e and f), those of PIGA^– cells were uniformly CD59^–. For the normal cells, 10,000 gated events were collected based on forward and side scatter and cells were analyzed for CD59 and CD45 or CD59 and DNA. Due to lower available cell numbers, for the PIGA-mutated and -nonmutated patient cell populations, about 1,000 gated events were collected.

Figure 3

Cell surface phenotypes of CD59^–CD34⁺ and CD59⁺CD34⁺ PNH cell populations prior to culturing. Each cell population was examined for CD38 (a and b), CD19 (c and d), CD64 (e and f), or CD71 (g and h) in three-color analyses as described in Methods. For each marker, a total of 20,000 gated events was collected based on forward and side scatter and cells were analyzed gating on CD34 positivity and CD59 positivity or negativity. In each instance about 75–100 events were recovered.

Figure 4

Expression of Fas receptor on PIGA^– and PIGA⁺ CD34 cells from four patients. Cells were stained for CD34, GPI-anchored CD59, and CD95 in three-color analyses. CD95 levels in patients’ nonmutated CD34⁺ cells (upper panels: a, c, e, and g) and patients’ PIGA-mutated CD34⁺ cells (lower panels: b, d, f, and h) are shown. As seen in the upper panels, CD95 levels on the nonmutated CD59⁺ cells were higher than on PIGA-mutated CD59^– cells in the same samples. For each patient, a total of 100,000 gated events was collected based on forward and side scatter and cells were analyzed gating on CD34 positivity and CD59 positivity or negativity. For each patient, about 200–400 events were recovered.

Figure 5

Heightened Fas-mediated killing of patients’ CD59⁺CD34⁺ cells in culture. (a) Separated PNH CD59^–CD34⁺ and CD59⁺CD34⁺ cells and normal CD59⁺CD34⁺ cells from cytopheresis samples of patient C and a healthy control were grown in culture as in Figure 2 in medium alone or medium containing agonist or antagonist mAb to CD95. The results represent the mean of six replicate experiments. (b) An identical experiment was done with separated cell populations from marrow samples of patient B and a corresponding control.