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TRANSPLANTATION
From the Department of Immunology, Weizmann Institute
of Science, Rehovot, Israel; Department of Hematology,
Kaplan Medical Center, Rehovot, Israel; Rabin Medical
Center, Petah Tikva, Israel; Bone Marrow Transplant, Hadassah
University Hospital, Jerusalem, Israel; and Universita
Degli Studi di Perugia, Ematologia E Immunologia Clinica, Perugia,
Italy.
Stem cell-dose escalation is one way to overcome immune rejection
of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was
addressed by the observation that cells within the human
CD34+ population are endowed with veto activity. In the
current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34+
cells for 7 to 12 days with an early-acting cytokine mixture including
Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the
expanded cells with fluorescence-activated cell-sorter scanning
revealed that the predominant phenotype of
CD34+CD33 The use of allogeneic hematopoietic stem cells for
the induction of transplantation tolerance as a prelude for subsequent organ transplantation or for adoptive cell therapy in cancer represents a difficult challenge. A major obstacle to achieving this desirable goal is associated with the elusive role of donor T cells. Although numerous studies demonstrated the valuable facilitating effect of T
cells on bone marrow engraftment,1-8 the risk of lethal graft-versus-host disease (GVHD) associated with alloreactive T cells
is unacceptable for organ transplantation or in the treatment of
nonmalignant diseases such as sickle cell anemia or autoimmunity.
Therefore, the development of new strategies for attaining durable
engraftment of allogeneic stem cells with minimal risk of GVHD in
patients pretreated with mild bone marrow ablative therapy is
desirable. One approach to overcoming immune rejection of incompatible
stem cells rigorously depleted of T cells has made use of stem
cell-dose escalation.9 However, although this modality
has become a clinical reality in the treatment of patients with
leukemia who have previously received intensive chemotherapy, it has
been suggested in studies in mice7 and nonhuman primates (X. Yao, unpublished data, July 2001) that the number of
hematopoietic precursors required to overcome the immune barrier in
recipients pretreated with sublethal regimens cannot be attained with
the state-of-the-art technology for stem cell mobilization.
Rachamim et al10 demonstrated that cells within the human
CD34+ population are endowed with potent veto activity. The
term "veto" relates to the ability of cells to neutralize cytotoxic
T-lymphocyte precursors (CTL-p) directed against their
antigens.11-17 Thus, when purified CD34+ cells
were added to bulk mixed lymphocyte reactions (MLRs), they suppressed
CTLs against matched stimulators but not against stimulators from a
third party.10
Clearly, the limitations of CD34+ collection in humans
might be overcome if it was possible to expand ex vivo the cells within the early progenitor population that are responsible for the veto effect. Our results in this study suggest that veto activity, unlike
the self-renewal potential associated with pluripotential stem cells,
can indeed be expanded in short-term culture of CD34+
cells. Thus, it was possible to harvest about 80-fold more veto cells
on culturing of CD34+ cells for 7 to 12 days under
conditions that induce predominantly myeloid differentiation. The
availability of such enriched veto cell preparations should provide a
new option for boosting engraftment of megadose CD34+
transplants in patients who have undergone mild conditioning treatment
and thereby induce durable immune tolerance without any risk of GVHD.
Collection of peripheral blood progenitor cells, processing,
and CD34+ cell purification
CTL reactivity
The primary MLR was prepared as follows. Responder lymphocytes from
donor C were allowed to react with stimulator cells from 2 donors (A
and B). Responder and stimulator donors were selected according to
their class I HLA typing to be non-cross-reactive with each other. The
cells from donor C (1 × 106/mL) were cultured with
1 × 106/mL irradiated (30 Gy) stimulator cells, with or
without addition of 0.5 × 106/mL CD34+ cells
(obtained from donor A). The cells were cultured in 6 mL complete
tissue culture medium (CTCM) and 10% fetal-calf serum (FCS; Biological
Industries, Kibbutz Beit Haemek, Israel) for 5 days. CTCM is RPMI 1640 containing 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL
streptomycin, 2 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, and 5 × 10 At the end of the primary MLR, cells were harvested from the MLR culture and separated by using the Ficoll method. Serial 4-fold dilutions of responder cells (from 4 × 104 to 0.156 × 103/well) were then prepared and seeded in round-bottomed, 96-well plates in 16 replicate samples for each dilution. Each well contained 105 irradiated stimulator cells from the donor who provided the original cells in the bulk MLR. The cultures were incubated for 7 days in CTCM, 10% FCS, and 10 U/mL recombinant interleukin 2 (IL-2; EuroCetus, Amsterdam, The Netherlands) in a final volume of 0.2 mL. Estimate of CTL activity Cytotoxic activity was assayed by transferring a fixed volume (100 µL) of limiting-dilution analysis cultures to conical-bottomed, 96-well plates (Greiner, Frickenhausen, Germany) and incubating the effector cells for 4 hours with 5 × 103 blasts (target cells) from the donor or a third party that had been stimulated with concanavalin A (Sigma, St Louis, MO) and labeled with chromium 51 (51Cr). The mean radioactivity from 16 replicate samples was calculated, and the percentage of specific lysis was determined by using the following equation: 100 × (experimental release spontaneous release)/(total release spontaneous
release). The release of 51Cr by target cells
cultured in medium alone was defined as spontaneous release; that
by cells lysed with 1% sodium dodecyl sulfate was defined as
total release.
Frequency calculation of CTL-p To calculate the frequency from the limiting-dilution culture readout, we used the following equation: ln y =fx + ln a
(which represents the zero-order term of the Poisson
distribution19), in which y is the percentage of
nonresponding cultures, x is the number of responding cells per
culture, f is the frequency of responding precursors, and a is the
y-intercept theoretically equal to 100%. Microwell cultures were
considered positive for a cytolytic response when values exceeded the
mean spontaneous release value by at least 3 SDs of the mean. The
percentage of responding cultures was defined by calculating the
percentage of positive cultures. The CTL-p frequency (f) and SE were
determined from the slope of the line drawn by using linear regression
analysis of the data. To evaluate the significance of the difference
between the slopes of 2 regression lines, t was calculated as follows: t = f1f2/ [SE2(f1) + SE2(f2)].20
The role of cell contact (transwell assay) Responder cells (1 × 106/mL) and irradiated stimulator cells (1 × 106/mL) were placed in the lower chamber of a transwell culture system (Transwell-col; Costar, Cambridge, MA). Purified donor CD34+ cells (0.5 × 106/mL) were placed together with the responder cells (1 × 106/mL) in the upper chamber. After 5 days of incubation, the cells were harvested and again cultured in limiting-dilution cultures.Flow cytometry analysis of intracellular interferon  (anti-IFN-) and
fluorescein isothiocyanate conjugated (PharMingen), or its appropriate
isotype control. Samples were analyzed by using a FACScan flow
cytometer, and data were analyzed with LYSIS II software (Becton
Dickinson, San Jose, CA).
Expansion culture Purified CD34+ cells (1 × 105/mL in a total volume of 1 mL) were cultured in 24-well plates in Iscoves modified Dulbecco medium containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 ng/mL recombinant human Flt3-ligand (FL), 50 ng/mL recombinant human stem cell factor (SCF), and 1 ng/mL recombinant human thrombopoietin (TPO; R&D systems, Minneapolis, MN). On day 5, the same doses of FL, SCF, and TPO were added; and on days 7 to 12, cells were harvested and tested for their veto activity.
The veto activity of noncultured CD34+ cells To evaluate whether veto cells can be expanded in a short-term culture of CD34+ cells, we first characterized the veto effect of noncultured CD34+ cells at different ratios of CD34+ to responder cells to define the lowest ratio at which veto activity was exhibited. As shown in Figure 1A, marked inhibition was detected at a CD34+-to-responder cell ratio of 0.5 or higher. At this ratio, 80% inhibition of the CTL activity was recorded when the culture contained stimulator cells from the CD34+ donor, whereas no significant inhibition was detected when the culture contained stimulators with a third-party origin. On the basis of this dose-response curve, all subsequent experiments were carried out at a CD34+-to-responder cell ratio of 0.5.
Figure 1B shows a summary of the results of 11 independent experiments
conducted with cells from different donors. HLA class I disparities for
the responder and stimulator cells used in each experiment are
summarized in Table 1. The average CTL
activity against target cells from the donor of the
CD34+ cells was 90.3% ± 20.8%, and it was markedly
inhibited by the addition of the CD34+ cells
(28.1% ± 24.7%). In contrast, the CTL activity against third-party
cells (100% ± 1%) was not affected significantly by the presence
of CD34+ cells (92% ± 12.5%). It could be
argued that if the addition of CD34+ cells to an
anti-third-party MLR led to a less extensive expansion of the
CD34+ cells compared with their expansion when added to an
anti-CD34+-donor MLR, the apparent reduction in CTL
activity might have resulted from a different dilution factor of the
CTL-p frequency by the CD34+ cells. However, in 11 experiments, the average cell recovery at the end of the 5-day culture
when comparing antidonor MLRs in the presence and absence of
CD34+ cells was increased by a factor of 1.35 ± 0.58,
whereas the increase in cell recovery in anti-third-party MLRs was
1.61 ± 0.65 (P
The veto specificity of the CD34+ cells was also exhibited
when the stimulators from the third party were added to the initial bulk MLR together with the stimulators from the CD34+ donor
(Figure 2). Thus, on addition of
CD34+ cells, the CTL-p frequency against the target cells
from the CD34+ donor was reduced by 1 log, from
10 × 10
The veto activity of CD34+ cells is not due to cold target inhibition It could be argued that the suppression observed on addition of CD34+ cells was due to cold target inhibition, that is, that the CD34+ cells added to the effector population could have competed with the 51Cr-labeled target cells for CTL-mediated lysis at the end of the culture period. Such competition will take place only when the CD34+ and target cells bear the same HLA class I antigens and therefore the observed chromium release will be reduced in antidonor cultures but not in anti-third-party cultures. To address this possibility, we isolated the effector T cells at the end of the bulk culture period by E-rosetting with sheep red blood cells (analysis of this fraction by flow cytometry showed more than 90% T cells and no detectable CD34+ cells) and tested the CTL activity of the purified T cells. As shown in Figure 3, the veto effect was retained after removal of the CD34+ cells.
Inhibition of antidonor response requires the addition of CD34+ cells in the first 24 hours of culture To study the critical period for the induction of the veto activity by CD34+ cells, we added the CD34+ cells at different times after initiation of the culture. As shown in Figure 4, inhibition of the response took place only when the CD34+ cells were added at initiation of the culture or 1 day afterward. No inhibition was detected when CD34+ cells were added on day 2 or later. Thus, the veto effect of CD34+ cells, similar to that of other veto cells,21-24 acts in the early induction phase of allogeneic CTLs.
The veto effect is not mediated by contaminating
CD34  cell fraction, which contains
a significant number of T cells. As shown in Table
2, although a marked reduction in CTL-p
frequency was found in MLRs to which purified CD34+ cells
were added, no inhibition was detected when cells of the CD34 fraction were added. Thus, the veto activity of
cells within the CD34+ cell fraction cannot be attributed
to residual CD34 cells. To investigate whether the
CD34+ veto activity could be delivered through the liquid
medium without cell contact, MLRs were prepared in transwell plates,
with 2 chambers separated by a membrane. The responder cells and the
irradiated donor stimulator cells were placed in the lower chamber and
the purified CD34+ cells were placed together with the
responder cells in the upper chamber. As shown in Table 2, CTL activity
was not reduced when the CD34+ cells were separated from
the stimulated responder cells by a membrane that may allow passage of
soluble factors but not cells.
Specific reduction by CD34+ cells of
effector cells positive for intracellular IFN- . Thus, in
7 experiments, the percentage of CD3+ cells expressing
IFN- on stimulation against cells obtained from the donor of the
CD34+ cells was reduced by 47.5% ± 15.3% when
CD34+ cells were added at a veto-to-responder cell ratio of
0.5. In contrast, when stimulated against third-party cells,
CD3+ cells positive for IFN- had a significantly lower
average inhibition level on addition of CD34+ cells
(18.7% ± 19.7%; P < .001).
Expansion of veto cells after short-term culture of CD34+ cells Expansion of human CD34+ cells in short-term cultures is usually associated with a significant loss of self-renewal capacity and with differentiation into myeloid phenotypes.25-29 To test whether CD34+ cells can be expanded ex vivo without loss of veto activity, CD34+ cells were cultured for a short period (7-12 days) in the presence of an early-acting cytokine mixture including FL, SCF, and TPO30 (concentrations of 50, 50, and 1 ng/mL, respectively). Phenotyping using fluorescence-activated cell-sorter scanning (FACS) revealed that although about 97% of the cells were CD34+CD33 before culture, the expanded cells
had 3 major subpopulations: CD34+CD33
(15.6%), CD34+CD33+ (33.0%), and
CD34CD33+ (50.5%; Figure
5). Furthermore, most of the cells
displayed CD13 (79%) and low CD4 levels (80%) and did not express
CD8, CD20, or CD56 (data not shown). Thus, as expected, the cells had
differentiated predominantly along the myeloid lineage.
The ex vivo-generated cells were tested for their effect on CTL-p
frequency (Figure 6) and activity (Table
3) and their effect on the levels of
effector T cells positive for intracellular IFN-
Finally, because human CD34+ cells exhibit a specific
inhibitory effect on the levels of effector T cells positive for
intracellular IFN-
The recognition that immune barriers can be overcome by cell-dose escalation with several nonalloreactive veto cells and other facilitating cells offers an attractive approach for the induction of durable hematopoietic chimerism.1-6,23,31-36 This approach was successfully implemented in the treatment of heavily pretreated patients with leukemia, allowing engraftment of 3-loci-mismatched, CD34+ stem cell transplants from haploidentical family members.37-39 Thus, the residual host immunity previously shown to be associated with a high rate of graft rejection (mediated predominantly by CTL-p40-42) in such transplants was overcome by using large doses of CD34+ cells. Subsequently, a study in a murine model using purified
Sca-1+Lin One possibility for addressing this issue was indicated by the
observation of Rachamim et al10 that cells within the
human CD34+ population are endowed with veto activity. In
the current study, we further characterized the veto activity of
CD34+ cells, showing different attributes of these cells,
including a dose-response curve, the time window in the MLR for
induction of the veto effect, and the role of cell contact. We also
ruled out, by removing the CD34+ cells at the end of the
MLR, the possibility of an artifact that might have been caused by cold
target inhibition. Thus, we fully confirmed and extended the early
observation that CD34+ cells possess marked veto activity.
In addition, we demonstrated that in parallel to the specific reduction
in CTL-p, CD34+ cells induce specific inhibition of the
levels of effector T cells positive for intracellular IFN- Considering that a large number of early myeloid progenitors can be expanded ex vivo by short-term cultures using different cytokine combinations,25-29,43-47 we hypothesized that if the veto activity could be retained along the myeloid differentiation pathway, then a small fraction of the CD34+ cells used for transplantation could provide a substantial source of veto cells. Our current results confirmed this hypothesis, demonstrating that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34+ cells for 7 to 12 days with an early-acting cytokine mixture including FL, SCF, and TPO. FACS analysis of the expanded cells revealed that the predominant
phenotype of CD34+CD33 The mechanism mediating the veto activity of different veto cells is still unknown. Veto activity was defined in 1980 by Muraoka and Miller11 as the capacity to specifically suppress CTL-p directed against antigens of the veto cells themselves but not against third-party antigens. Interestingly, it has been shown that some of the most potent veto cells are of T-cell origin; in particular, a very strong veto activity was documented for CD8+ CTL lines or clones.2,12-15,48-51 The specificity of the veto effect mediated by CTL clones was shown in several studies to be unrelated to their T-cell-receptor specificity.16,52,53 Although early studies emphasized the role of CD8-mediated apoptosis,16,48,54,55 more recent evidence indicated a role for Fas-FasL.56,57 Moreover, it was demonstrated by using blocking anti-CD8, by generating CTLs from FasL or perforin-mutated mice, and by gene transfer of FasL, that the veto activity of nonalloreactive CD8+ CTLs is dependent on the simultaneous expression of both CD8 and FasL.24 Preliminary data suggested that the veto activity of human
CD34+ cells is also mediated by apoptosis (H.G., et al,
unpublished data, January 2002); however, it seems that
neither CD8 nor FasL is a likely mediator of this activity. In
addition, Sca-1+Lin Regardless of the mechanism involved, our new observation that
CD33+ cells derived on short-term culture from
CD34+CD33 Our current results, which suggest that ex vivo expansion of CD34+ cells can afford about 28- to 80-fold more veto cells before the day of transplantation, could be applicable to achievement of tolerance under less intensive conditioning protocols. Currently, conventional unseparated bone marrow or peripheral blood stem cell transplants are used extensively after a nonmyeloablative conditioning in HLA-identical recipients with leukemia.58-60 These transplants are associated with significant alloreactivity because of the large number of T cells in the transplant inoculum that, in most instances, eradicate hematopoiesis in the recipient. This alloreactivity is also associated with a marked incidence of lethal GVHD. Therefore, these "minitransplants" are limited to use in patients with end-stage hematologic malignant diseases for whom a perfectly HLA-matched donor is available. The significant risk of GVHD precludes the use of nonmyeloablative hematopoietic transplants in their current form for induction of transplantation tolerance before organ transplantation or in the treatment of other, nonmalignant diseases. The availability of novel sources of veto cells such as those provided in this study by ex vivo expansion of CD34+ cells should eliminate the risk of GVHD and might contribute to the clinical realization of the important goal of tolerance induction after nonmyeloablative conditioning.
Submitted April 23, 2001; accepted January 23, 2002.
Supported in part by a grant from the Gabrielle Rich Leukemia Research Foundation. Y.R. is the incumbent of the Henry Drake Professorial Chair.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yair Reisner, Weizmann Institute of Science, Department of Immunology, Rehovot, 76100, Israel; e-mail: yair.reisner{at}weizmann.ac.il.
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Top
Abstract
Introduction
) and a third-party donor (
) were
cocultured for 5 days. Different numbers of purified CD34+
cells were added to obtain the indicated ratios of CD34+ to
responder cells. The responder cells were then cultured again for 7 days under limiting dilution, and the CTL activity was determined by
51Cr-release assay. Two experiments were carried out. Data
are from 1 representative experiment and represent the percentage of
specific lysis at a cell concentration of 4 × 104
effector cells/well. The asterisk indicates a significant difference
(P < .001 on t test compared with control
cultures without CD34+ cells). (B) The average CTL response
(±SD) in the presence (
.13).
) and a third party (
)
for 5 days in the absence (
,
) of
CD34+ cells added at a veto-to-responder ratio of 0.4:1.
The responder cells were then divided equally, and each fraction was
cultured again for 7 days under limiting dilution with stimulators from
the donor of the CD34+ cells (A) or stimulators from the
third-party donor (B). The CTL activity was determined by
51Cr-release assay with the relevant target cells. The
CTL-p frequency was calculated. The parameters for the different
regression lines were as follows. (A) CTL-p frequency against
stimulators from the CD34+ donor in the absence of
CD34+ cells (
3 domain of class I MHC.
Science.
1991;252:1424-1427
(IL-1