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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2189-2196
By
From the Divisione di Ematologia e Centro Trasfusionale, Ospedali
Riuniti Bergamo, Bergamo, Italy; and Clinica Pediatrica
Università di Milano, Monza, Italy.
A major obstacle in purifying either autologous or allogeneic
hematopoietic stem cells from granulocyte colony-stimulating factor
(G-CSF) mobilized circulating progenitor cells (CPC) is represented by
the huge cellularity present in each apheretic product. To obtain a
significant debulking of unwanted cells from the leukapheresis, we
developed a modified protocol of immune rosetting whereby human
ABO-Rh- compatible red blood cells (RBCs) are treated with chromium
chloride and then coated with murine monoclonal antibodies (MoAbs)
against leukocyte antigens. When experiments were performed with
leukaphereses obtained from normal donors or from T-cell acute
lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine
MoAbs against human mature myeloid cells (CD11b) and T cells (CD6);
whereas, in the case of patients with B-precursor ALL, B-cell
non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were
coated with anti-CD11b only. After incubation with CPC, rosetting cells
(myeloid precursor cells, granulocytes, monocytes, and T cells) were
removed by Ficoll-Hypaque density gradient centrifugation with a blood
cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a
significant reduction of the initial cellularity was consistently
obtained (range, 72% to 97%), whereas the median absolute recovery of
the CD34+ cells was above 85% (range, 64 to 100), with a
10-fold relative enrichment ranging from 3% to 41%. In a second step,
CPC can be further purged of contaminating T or B cells by incubation
with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a
SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to
four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+
cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At
the end of the procedure the T- or B-cell depleted fraction retained a
high proportion of the initial hematopoietic CD34+ stem
cells, with a median recovery above 70% (range 48% to 100%) and an
unmodified clonogenic potential. In five patients (two follicular NHL
and three ALL) the purified fraction of stem cells was found disease
free at the molecular level as assessed by polymerase chain reaction
(PCR) analysis of the t(14;18) chromosome translocation or
clono-specific DNA sequences of IgH or T-cell receptor
IN THE NORMAL bone marrow the
CD34+ cell fraction contains virtually all the
hematopoietic progenitor cells (HPCs).1 After priming with
hematopoietic growth factors (G-CSF or GM-CSF), either alone or in
combination with high-dose chemotherapy, mobilized circulating
progenitor cells (CPCs) expressing the CD34 antigen on the cell surface
can be easily collected from peripheral blood in quantities
approximately 10-fold higher than previously obtained from bone
marrow.2 The purification of human stem cells has clinical
relevance for autologous3 and allogeneic
transplantation4 and for the usage of the stem cells as
vehicles for gene transfer.5,6 High-dose chemotherapy and
autologous transplantation are increasingly being used to treat
patients with hematologic malignancies and solid tumors even though the
neoplastic contamination of CPCs7-9 may contribute to
subsequent relapses.10 Because many tumor cell types
including multiple myeloma (MM), lymphomas, breast and ovarian cancer,
and neuroblastoma do not usually express the CD34 antigen,
CD34+ positive selection may be effective for the purging
of the autologous grafts. Different commercial devices are now
available for laboratory and clinical scale enrichment of these cells
by immunoaffinity columns or immonomagnetic beads, and preliminary
clinical results have already been obtained by using purified
CD34+ cells for autologous
transplantation.8,9,11-15 More recently, some investigators
started to analyze the feasibility of using purified CD34+
cells for allogeneic transplantation.4,16,17 In fact,
allogeneic CPC preparations may provide the possibility to obtain
T-cell depleted fractions of stem cells while preventing an
unacceptable loss of hematopoietic progenitors. Therefore,
CD34+ cell selection can be used as an efficient method for
preventing graft versus host disease (GVHD) without an increased risk
of graft failure or rejection.18 Unfortunately, at least
two major concerns are opposing a wide clinical use of purified
CD34+ cells. The immune reconstitution can be severely
compromised if inadequate numbers of T and B cells are present in the
graft, a problem that deserves particular attention, especially in the case of patients with nonhematologic malignancies. Moreover, most acute
leukemia cell types express the CD34 antigen, thus preventing the use
of this method of purification in the autologous setting.
The aim of this study was to develop an efficient, reproducible, and
relatively inexpensive method for clinical scale preparation of CPC for
transplantation procedures. Furthermore, the selective- and
lineage-specific elimination of the neoplastic fraction from the
autologous graft as well as the normal T cells from the allogeneic graft was the aim of the same procedure. This novel methodology consists of two distinct steps: in the first step, 85% of the initial
cellularity (granulocytes, monocytes, and eventually T cells) is
allowed to form rosettes with chromium chloride-treated human
ABO-Rh-compatible red blood cells (RBCs) coated with murine monoclonal
antibodies (MoAbs) antihuman CD11b (and CD6 when T-cell depletion is
needed) and then removed by gradient sedimentation, without a
significant loss of the CD34+ cells present in the input.
Subsequently, in step two, this debulked apheresis is very efficiently
purged of unwanted B or T cells by the use of lineage-specific
monoclonal microbeads.
Mobilization and harvesting of autologous or allogeneic CPCs.
Autologous CPCs were collected in patients with non-Hodgkin's lymphoma
(NHL), acute lymphoblastic leukemia (ALL), and MM, after different
consolidation protocols of high-dose chemotherapy followed by rhG-CSF
(Filgrastim; Roche, Milan, Italy) administration (5 µcg/kg/day), as previously described.19 Leukapheresis was performed as soon as white blood cells (WBCs) were at least 3.0 × 109/L and peripheral blood CD34+ cells were Immune rosettes.
The method is based on a previously published procedure based on the
ability to couple murine MoAbs to RBCs by chromium
chloride.21 The following mouse hybridoma cell lines were
obtained from American Type Culture Collection (ATCC, Rockville, MD):
OKM1 (IgG2b, ATCC CRL 8026) reactive with human granulocytes,
monocytes, NK cells and committed myeloid precursor cells (CD11b
antigen); and T12 (IgM, clone 3Pt12B8; ATCC HB8136) reacting with human
T cells and some B cells (CD6 antigen). Partially purified preparations of these MoAbs were obtained by ammonium sulphate precipitation of
spent culture supernatants of each hybridoma. ABO-Rh-compatible, irradiated (25 Gy), and filtered human red blood cells (HRBCs) were
obtained from the Blood Bank. For coating with MoAbs, 150 mL of packed
HRBCs were washed three times in normal saline (centrifugation at 3000 RPM for 5 minutes at room temperature) with a COBE 2991 apparatus and a
blood cell processor set. After the third wash, 30 mL (3 mg/mL) of
partially purified MoAbs (CD11b and eventually CD6 in the case of
normal donors and T-ALL patients) were added at the same blood
processor set. Thereafter, and under continuous agitation, 250 mL of a
0.1% solution of chromium chloride (CrCl3 .6H2O; Sigma, St.Louis, MO; prepared in normal saline from
a 1% [W/V] stock solution with the pH adjusted to 5.0 with 10 N
NaOH) were added dropwise over a 15-minute time period. After
incubation for 5 additional minutes at room temperature the reaction
was stopped by the addition of 300 mL of phosphate-buffered saline (PBS) supplemented with 2.5% Human Serum Albumin (PBS-HSA).
Apparently, the isotype of the antibodies (IgM or IgG) does not affect
the coupling process to RBCs. MoAb-coated HRBCs were washed twice, resuspended in 100 mL of PBS 2.5% HSA, and mixed with 11 to 58 × 109 white blood cells (200 mL final volume, hematocrit less
than 5%) obtained from leukaphereses of G-CSF-treated individuals. Rosette formation was performed within the same blood cell processor set by two centrifugation steps at 3000 RPM for 30 minutes. At the end
cells were resuspended in 400 mL of PBS 2.5% HSA, transferred, and
layered onto the top of 200 mL Ficoll Hypaque (by using a second blood
cell processor set), and centrifuged for 45 minutes at 3000 rpm.
Nonrosetting cells were harvested at the Ficoll interface and washed
twice with PBS-HSA. After 5 minutes of incubation with hypotonic
NH4 Cl buffer (NH4 Cl 8.99 gr/L,
KHCO3 1gr/L, Na4 EDTA 0.037 gr/L, pH 7.3) to
lyse residual erythrocytes, cells were washed with PBS-HSA, resuspended
in RPMI 1640 10% FCS, counted, and stained with MoAbs for FACS
analysis. The described procedure required approximately 3 hours of
work performed by one operator and the estimated cost (including
production and purification of antibodies, chemical reagents, tissue
culture media, two blood cell separation sets, and 1 U of filtered,
irradiated, RBCs) was 400 US dollars.
Immunomagnetic purging of T or B cells.
To obtain a high degree of T- or B-cell depletion, partially purified
hematopoietic progenitors (0.5 to 13 × 109 cells)
obtained by immune rosetting were labelled with primary unconjugated
MoAbs reacting against T cells (anti-CD2 antigen, clone 35.1; IgG2a,
ATCC HB222; and anti-CD7, clone WT1, IgG1) or with a panB MoAb (clone
HD37, anti CD19, IgG1; kindly provided by Dr. Moldenhauer, Heidelberg,
Germany). In MM patients (two cases), cells were incubated with
anti-CD19 and anti-CD5622 (clone N901, IgG1;
kindly provided by Dr JD Griffin, Boston, MA). Thereafter, an indirect
labelling was performed with goat antimouse magnetic microbeads,
according to the manufacturer instructions (Miltenyi Biotec,
Germany). After labelling, cells were washed as above, resuspended in
PBS-HSA (30 mL final volume), and layered, by the use of a peristaltic
pump, onto the top of a type-D depletion column (composed of
ferromagnetic fibers) inserted within a SuperMACS apparatus (Miltenyi
Biotec). Unstained cells were eluted with PBS-HSA from the column kept
within the magnetic field of the cell separator. The T- or B-cell
depleted fraction of HPCs was finally resuspended in autologous plasma,
counted, phenotypically analyzed, and cryopreserved. The time required
to complete this negative depletion was 3 hours and the estimated cost
of two vials of goat antimouse magnetic microbeads and one type-D
depletion column, necessary for each procedure, was 1,500 US dollars.
Molecular evaluation of minimal residual disease (MRD).
In patients with ALL, MRD was evaluated by molecular analysis of
junctional regions of rearranged T-cell receptor (TCR) Effect of immune rosetting on overall recovery and composition of G-CSF
mobilized CPC.
In normal donors receiving G-CSF (10 µg/kg/d) for 5 to 6 days, the procedure of CPC mobilization and collection was safe and always well tolerated. The apheretic products were debulked of committed myeloid cells and T lymphocytes by immune rosetting with
anti-CD11b and -CD6 antibodies. The experimental procedure of immune
rosetting was always carried out on the second or third harvest after a
sufficient amount of unmodified CPCs were used or stored in liquid
nitrogen. As shown in Table 1, 18 experiments were performed with a median starting cellularity of 41.3 × 109 (range, 29.7 to 58.2) nucleated cells. After
immune rosetting, the median reduction of the initial cellularity was
91% (range, 72 to 97) and the cell loss was mainly due to a marked
(more than 85%) depletion of myeloid cells and T lymphocytes. A
parallel enrichment of CD34+ cells was obtained (more than
10-fold) with a median recovery of HPCs of 92% (range, 67 to 100).
Similar results were obtained by using autologous CPCs collected in
T-ALL patients after high-dose chemotherapy and G-CSF administration.
Due to the effect of high-dose chemotherapy, the starting cellularity,
as well as the absolute number of CD3+ cells, was lower
than that observed in the apheresis obtained from normal allogeneic
donors, whereas the amount of CD34+ cells was significantly
higher (Table 1). Nonetheless, the debulking effect of the procedure
(88% overall cell loss, with more than 84% of mean CD3+
cell loss), as well as the absolute recovery and the enrichment fold of
CD34+ cells, was remarkably similar. In patients with B
precursor ALL and B-NHL the apheretic products were debulked by immune
rosetting with RBCs coated with CD11b only. Also, in this case, the
initial cellularity and the absolute number of T and B lymphocytes were less abundant as compared to normal donors. As expected, the loss of
CD3+ cells was lower, even though the overall reduction
obtained by immune rosettes was similar.
Highly efficient depletion of T cells by anti-CD2-CD7 or B cells by
anti-CD19 magnetic microbeads.
To purge the contaminating normal or neoplastic T lymphocytes still
present in the apheretic products after the debulking procedure with
immune rosettes, partially purified allogeneic (9 experiments from
normal donors) or autologous (7 experiments from T-ALL patients) CPCs
were incubated with a mixture of anti-CD2 and -CD7 MoAbs and indirectly
stained with goat antimouse magnetic microbeads. After loading onto a
D-type depletion column, normal or leukemic contaminating T cells were
significantly removed as judged by staining with anti-CD3 MoAbs
(Table 2) and other T-cell-specific antigens like CD5, CD4, and CD8 (data not shown). The overall T-cell
depletion obtained by the two combined procedures allowed a final 3 to
4 logs reduction of the T-cell content. Despite such aggressive removal
of T cells, the median overall recovery of CD34+ cells was
above 70% in both autologous and allogeneic CPCs (Table 2). Similar
experiments were performed to remove B cells in CPCs obtained from
patients with B-precursor ALL, B-NHL, and MM. As shown in
Table 3, the percent and the absolute
number of CD19+ cells detectable after this purification
approach were very limited. Again, the absolute recovery of
CD34+ cells from the initial leukapheresis was excellent,
with a mean value above 80%.
Evaluation of MRD.
A PCR-based quantitation of MRD was performed on CPC samples obtained
from five patients before and after depletion of contaminating tumor B
or T lymphocytes by purging with lineage-specific immunobeads. Molecular analysis was performed by demonstration of chimeric genomic
products generated by the t(14;18) chromosomal translocation or by
analysis of leukemia-specific DNA sequence, amplified from the
rearranged TCR
Transplantation of T- or B-cell purged autologous and allogeneic
CPC.
We performed autologous transplantation in three patients by using CPCs
highly purified by immune rosetting and immunomagnetic purging. In the
case of the T-ALL patient presented in Fig 1, after a myeloablative
therapy with high-dose Ara-C (2 g/m2 × 2/d for 6 days) and fractionated total body irradiation (TBI, 12cGy), the
purified, leukemia-free fraction of HPCs obtained at the end of the
purification procedure (6.8 × 106 /kg
CD34+ cells) was autografted. The neutrophil engraftment
(more than 1.5 × 109/L) was observed after 12 days
and more than 20 and 100 × 109 /L platelets were
counted at days +15 and + 30, respectively. Interestingly, the
molecular evaluation of the bone marrow 100 days after transplantation
did not show persistence of leukemic cells within the patient (data not
shown). Similar results were obtained in two MM patients resistant to
conventional chemotherapy and who underwent transplantation after a
high-dose Melphalan (200 mg/m2) conditioning regimen, with
CD19 and CD56 purged stem cells.26,27 The numbers of
infused CD34+ cells were 8.3 and 10 × 10 6 /kg CD34+ cells, respectively. A rapid
hematologic engraftment was observed in both patients, because more
than 1.5 × 109/L neutrophils were counted at days +10
and +11 and more than 20 × 109/L platelets were
counted at day +14 (Table 5).
The manipulation of specific cell subpopulations of marrow or
peripheral blood origin has become an interesting way to increase the
applicability and reduce the toxicity of hematopoietic transplantation. However, the manipulation of CPCs obtained from G-CSF treated normal
donors or patients is hampered by the huge cellularity present in the
apheretic products, which often exceeds the absolute number of 50 × 109 cells, and by the fact that under the
stimulatory effect of G-CSF, mature myeloid cells (mostly granulocytes)
acquire different characteristics of cell density preventing their
sedimentation on normal Ficoll gradients. Therefore, two main
approaches to the CD34+ purification have been taken either
by positive or by negative selection. Indeed, in the perspective of
genetic manipulation, positive selection of CD34+ cells is
likely to be the ideal option to ensure that the selected gene is
transduced only in the small target population of pluripotent progenitor cells.32 Although transplantation of positively
selected CD34+ cells purified by immunoaffinity columns or
immunomagnetic bead adsorption has been shown as a feasible
procedure,4,9,11-15 the specific depletion of unwanted
cells seems preferable for several reasons including the preservation
of the manipulation of CD34+ cells from binding with murine
MoAbs and the need of their subsequent detachment by using chemical or
physical methods. Moreover, most acute leukemias of both myeloid and
lymphoid origin are positive for CD34 antigen expression thus reducing
in this setting the clinical applicability of methods that rely only on
the positive selection of the stem cell fraction. On the contrary, the
selective elimination of residual neoplastic cells detectable in the
stem cell fraction obtained from some patients with MM,33
follicular lymphoma,34 and breast cancer35
could be achieved by the use of MoAbs either in association with
complement,36 conjugated to toxic compounds,37
or by the use of magnetic microbeads proven to eradicate the neoplastic
clone at the molecular level.25,38 However, it has to be
mentioned that positive selection of CD34+ cells, combined
with negative depletion steps, could also achieve high levels of
purity,39 even though purging strategies based only on
depletion techniques could avoid an extensive removal of T cells from
the autograft of patients with B-lymphoproliferative disorders and
solid tumors, thus reducing the risk of long-lasting immunodeficiency.
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Abstract
Introduction
and 
chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a
highly efficient purging of T or B cells from G-CSF expanded and
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neoplastic clone in the autologous stem cell inoculum as well as for
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Abstract
0.5%. Ten liters of blood were processed daily through an indwelling
central venous catheter (Groshong CV Catheter; Band Inc, Salt Lake
City, UT) by using a cell separator COBE spectra (COBE, Lakewood, CO).
Allogeneic CPCs were collected from HLA-identical, MLC-negative normal
siblings upon treatment with 2 × 5 µg/kg/day of rhG-CSF
administered subcutaneously every 12 hours for 5 or 6 consecutive days.
The procedure of leukapheresis was started on day 5 (after the ninth
dose of G-CSF) by using a COBE spectra cell separator. Ten liters of
blood were processed daily by using the cubital vein in all donors, as
previously described.