|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3212-3221
Induction of Donor-Type Chimerism and Transplantation Tolerance Across
Major Histocompatibility Barriers in Sublethally Irradiated Mice by
Sca-1+Lin Bone Marrow Progenitor
Cells: Synergism With Non-Alloreactive (Host × Donor)F1 T Cells
By
Esther Bachar-Lustig,
Hong Wei Li,
Hilit Gur,
Rita Krauthgamer,
Hadar Marcus, and
Yair Reisner
From the Department of Immunology, Weizmann Institute of Science,
Rehovot, Israel.
 |
ABSTRACT |
Induction of transplantation tolerance by means of bone marrow (BM)
transplantation could become a reality if it was possible to achieve
engraftment of hematopoietic stem cells under nonlethal preparatory
cytoreduction of the recipient. To that end, BM facilitating cells,
veto cells, or other tolerance-inducing cells, have been extensively
studied. In the present study, we show that BM cells within the
Sca-1+Lin cell fraction, previously shown
to be enriched for early hematopoietic progenitors, are capable of
reducing specifically antidonor CTL-p frequency in vitro and in vivo,
and of inducing split chimerism in sublethally 7-Gy-irradiated
recipient mice across major histocompatibility complex barriers. The
immune tolerance induced by the Sca-1+Lin
cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host
immunity remaining after 7 Gy total body irradiation is very large and,
therefore, it may be very difficult to harvest sufficient cells for
patients. This challenge was further addressed in our study by
demonstrating that non-alloreactive (host × donor)F1 T
cells, previously shown to enhance T-cell-depleted BM allografts in
lethally irradiated mice, synergize with
Sca-1+Lin cells in their capacity to
overcome the major transplantation barrier presented by the sublethal
mouse model.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE INDUCTION of substantial and durable
hematopoietic chimerism without graft-versus-host disease (GVHD),
following minimal conditioning of the recipients, represents an
extremely desirable goal in transplantation biology, because it is
generally associated in murine models with a permanent transplantation
tolerance.1-4 It has been shown that large doses
of T-cell-depleted bone marrow (BM) transplants can
permanently paralyze the resistance of host-type T cells remaining
after exposure to sublethal 5.5 to 7.5 Gy total body irradiation
(TBI).5 Subsequently, similar results were also obtained
when the preparation of the recipients was based on treatment with
anti-T-cell antibody plus thymic irradiation.6 It was
shown in these studies that residual host-type cytotoxic T-lymphocyte
precursors (CTL-p), surviving the sublethal conditioning, were markedly
abrogated by the megadose BM transplants. This effect could be
attributed to several types of accessory cells in the BM, as previously
shown in murine models using lethally irradiated recipients7-14 or by in vitro studies measuring veto
activity of different mouse15 or monkey16 BM
cell subpopulations. A common characteristic of most, although not all,
facilitating cells described in the literature is their expression of
CD8 molecules on the cell surface. Although different facilitation
mechanisms might be associated with different CD8+ cells,
studies suggest that the CD8 molecule itself might be directly
associated with this effect. Sambhara et al,15 using specific antibodies, showed that the interaction of CD8 molecules on
the veto cells with the  3 domain of H-2 class 1 molecules on the
effector cells can induce apoptosis specifically in the effector CTL-p,
directed against class I antigens of the veto cells. Furthermore,
attachment of soluble CD8 to the cell surface of nonveto cells, such as
fibroblasts, has endowed these cells with a potent veto
activity.17 Altogether, these observations have led to a
particular emphasis on BM facilitation mediated by CD8+ cells.
Most recently, with the advent of granulocyte colony-stimulating factor
(G-CSF) stem cell mobilization in humans, it has been shown that the
high rate of graft rejection experienced in the past in leukemic
recipients of T-cell-depleted HLA disparate BM transplants can now be
overcome by using large doses of stem cells that are collected from the
donor's blood after G-CSF mobilization.18-20 These
clinical results indicated that cells within the CD34-enriched transplants are capable of overcoming the host resistance, and very
recently Rachamim et al21,22 further supported this
hypothesis by demonstrating in vitro that cells within the CD34 cell
fraction possess marked veto activity.
Based on these indications, we attempted to investigate, in the mouse
model, whether cells within the hematopoietic progenitor BM population
are endowed with tolerance-inducing activity and are capable of
overcoming the marked resistance, typical of sublethally irradiated
allogeneic recipients. To address this question, we purified in the
present study different BM cell fractions and we tested their capacity
to induce split chimerism as well as to specifically reduce antidonor
CTL-p, both in vitro and in vivo. We found that early hematopoietic
Sca-1+Lin cells, depleted of
CD8+ cells, exhibit in vitro similar veto activity to that
shown for human CD34 cells and, moreover, cells within the
Sca-1+Lin cell fraction are
indeed capable of overcoming the marked immunoresistance of the host.
However, these studies also indicate that the cell number required for
tolerance induction in sublethally irradiated recipients is rather
large and it may be difficult to collect a sufficient quantity in
humans. Therefore, we explored other cells that might synergize with
the Sca-1+Lin cells in overcoming the
challenge of the marked host immunity typical of the sublethal mouse
model. To that end, the potential of (host × donor)F1
non-alloreactive T cells, previously shown23 to enhance
engraftment of T-cell-depleted BM allografts in lethally irradiated
recipients, was evaluated.
| |
MATERIALS AND METHODS |
Animals.
Mice used were female 6- to 12-week-old C3H/HeJ and 4- to 6-week-old
BALB/c, C57BL/6, C57BL/Beige, and
(C3H/HeJ×C57BL/6)F1, obtained from
the Roscoe B. Jackson Memorial Laboratory (Bar Harbor, ME) or the
Weizmann Institute Animal Breeding Center (Rehovot, Israel). All mice
were kept in small cages (5 animals in each cage) and fed sterile food
and acid water containing cyprofloxacin (20 µg/mL).
Purification of murine stem cells by magnetic sorting.
BM cells were prepared as previously described,24 and then
subjected to a stem cell purification procedure based on their expression of stem cell antigen-1 (Sca-1)25 and the lack of expression of cell-surface antigens associated with differentiated hematopoietic cell lineages typical for B cells (CD45/B220),
myelomonocytic cells (CD11b/mac-1), and T cells (CD4/L3T4, CD8/Ly-2).
Sca-1-positive, lineage-negative cells
(Sca-1+Lin) were purified from 6 to 10 × 109 BM cells (obtained from 100 C57BL/6 donors 5 to
9 weeks old, respectively), which were initially
enriched for mononuclear cells by separation on Ficoll-Paque Plus
(Pharmacia Biotech AB, Uppsala, Sweden). BM cells (50 × 107/20 mL) were layered on Ficoll (30 mL) in 50-mL Falcon
tubes (Becton Dickinson, San Jose, CA). The cells were centrifuged at
room temperature, at 800g for 30 minutes, and the cells in the
Ficoll interface were collected. This cell fraction was magnetically
labeled with anti-Sca-1 antibodies conjugated to microbeads using a
multi-parameter magnetic cell sorting (MACS) Sca-1 Multisort Kit
(Miltenyi Biotec, Bergisch Gladbach, Germany).
Sca-1+-labeled cells were then purified by a positive
selection column in a magnetic field. The microbeads were then removed
from the Sca-1+ cells using Multisort release reagent, to
allow subsequent magnetic labeling and separation of Sca-1+
cells according to expression of lineage markers. The depletion of
Sca-1+ cells expressing lineage markers was performed by
positive selection following labeling of Sca-1+ cells with
MACS microbeads conjugated to antibodies directed against CD45/B220,
CD8/Ly-2, CD4/L3T4, CD11b/mac-1, and anti-NK (DX5), when natural killer
(NK) cells were depleted. The negative and positive fractions of this
separation, the Sca-1+Lin and
Sca-1+Lin+ cell fractions, were collected. In a
typical experiment starting with 6 × 109 BM cells,
cell recovery after Ficoll_fractionation and in the Sca-1+, Sca-1+Lin+, or in the
Sca-1+Lin cell fraction was 2 × 109, 1 × 108, 71.5 × 106, and 5 × 106, respectively.
Cytofluorimetric analysis of the fractionated cells was performed by
double-immunofluorescent staining, using the following directly labeled
antibodies (obtained from Pharmingen San Diego, CA): fluorescien
isothiocyanate (FITC)-Sca-1/Ly-6A/E (clone D7), R-phycoerythrin
(PE)-CD11b (clone M1/70), PE-CD8a/ Ly-2 (clone 53-6.7), PE-CD45/B220
(clone RA3-6B2), PE-CD4/L3T4 (clone RM-4-5), and PE-pan-NK cells (clone
DX5) for NK cell detection. Nonspecific staining was analyzed with rat
Ig isotype controls: FITC-rat IgG2a, PE-rat
IgG2a, and PE-rat IgG2b.
Preparation of non-alloreactive F1 T cells.
Thymocytes from (C3H/HeJ×C57BL/6)F1 mice were
separated by differential agglutination with peanut agglutinin into
mature and immature fractions as described.26
Irradiation and BM transplantation.
Mice were exposed to a single dose of 7 Gy (sublethal conditioning) or
10 Gy (lethal conditioning) TBI from a Gamma beam 150-A 60Co source (produced by the Atomic Energy of Canada,
Kanata, Ontario, Canada) with focal skin distance of 75 cm, at a 0.65 Gy/min dose rate. The following day the mice received, intravenously,
BM subpopulations as described in Results. In the experiments studying
the facilitating effect of non-alloreactive T cells, mice were injected
with Sca-1+Lin cells supplemented with
F1 T cells.
Chimerism analysis.
Chimerism was determined 30 days posttransplant by cytofluorimetry.
Mice were bled from the retro-orbital vein using heparin-coated glass
capillaries. Peripheral blood cells were fractionated on Ficoll-Paque
plus, and the isolated mononuclear cells of each mouse were
triple-stained by direct immunofluorescence, with FITC anti-H2b monoclonal antibody specific for the donor, PE
anti-H2k for host-type major histocompatibility complex
(MHC) antigens, and Cy-chrome-anti-CD3 antibody. The following
antibodies were obtained from Pharmingen (San Diego, CA):
FITC-H-2Dd (clone 34-2-12), PE-H-2Kk (clone
36-7-5), FITC-H-2Kb (clone AF6-88.5), and Cy-Chrome-CD3e
(clone: 145-2C11).
Skin grafting.
Skin grafting was performed as previously described,27 with
one additional step. Briefly, a circular piece of skin was removed from
the recipient mouse and replaced by skin taken from the donor's trunk
after it had been cleaned of fat and connective tissue. The attachment
of the skin graft to the recipient was achieved by spraying several
thin layers of acrylic plastic spray (Nobecutan Spray; Astra,
Sodertalje, Sweden) without any accessories. After the plastic dried (2 minutes), several layers of antibiotics (Rikospray antibiotic; Riker
Laboratories, Loughborough, UK) were sprayed on the graft, and the mice
were then transferred separately into individual small cages in which
they were kept throughout the observation period. The transplanted skin
grafts were observed daily and the graft status was recorded, with an
acceptance score based on size, color, contraction, and hair growth of
the graft. Rejection started with the loss of hair and culminated in
necrosis of the graft epithelium that was occasionally associated with ulcer formation. Definite rejection was taken as the time of complete sloughing, or when a dry scab was formed.
Veto activity of Sca-1+Lin cells.
To determine whether mouse Sca-1+Lin
cells possess veto activity, spleen cells from C3H/HeJ mice (2 × 106/mL) were incubated for 5 days with irradiated (25 Gy)
allogeneic spleen cells (1 × 106/mL) from C57BL/6
(stem-cell matched) or BALB/c (third party) mice. C57BL/6
Sca-1+Lin or Sca-1
cells were added to the primary MLC at a 0.5:1 veto:responder cell
ratio. The responder cells were then recultured under limiting dilution
conditions for 7 days. The CTL-p activity was determined by
51Cr-release assay.
Direct limiting dilution culture of CTL-p.
Responder cells of the veto activity cultures or splenocytes of C3H/HeJ
chimeric mice (irradiated by 7 Gy TBI and transplanted with 2 × 105 Sca-1+Lin cells from
C57BL/6 donors) were fractionated on Ficoll-Paque plus and the isolated
mononuclear cells were plated in a limiting dilution culture. Responder
cells (40 to 0.16 × 103) were cultured for 7 to 8 days in round-bottomed 96-well plates (Nunc, Roskilde, Denmark) in 16 replicates, in the presence of 106 cells/well of irradiated
(20 Gy) allogeneic (C57BL/6, BALB/c) or syngeneic (C3H/HeJ) splenocytes
and human recombinant interleukin-2 (10 U/mL; Eurocetus, Milan, Italy)
in a final volume of 0.2 mL complete tissue culture medium (CTCM) at
37°C in a 10% CO2 air incubator. CTCM is RPMI 1640 which contains 2 mmol/L L-glutamine, 100 U/mL penicillin, 0.1 mg/mL
streptomycin, 2 mmol/L HEPES, 1 mmol/L sodium pyruvate, 0.1 mmol/L
nonessential amino acids, and 5 × 105 mol/L
2-mercaptoethanol, supplemented with 10% fetal calf serum (Biological
Industries, Kibbutz Beit Hemek, Israel).
Estimate of cytotoxic cell frequency.
Splenocytes were harvested from individual limiting dilution culture
wells and were assayed for cytotoxic activity by transferring a fixed
volume (100 mL) to conical-bottomed 96-well plates (Greiner, Frickenhausen, Germany) containing 5 × 103
51Cr-labeled Concanavalin A (Sigma, St Louis, MO) blasts of
C3H/HeJ, C57BL/6, or BALB/c, respectively, as target
cells. As described by Ryser and
MacDonald,28 microwells were considered positive for
cytolytic activity when they exceeded the mean spontaneous release
value (determined in a group of parallel wells that contained irradiated stimulating cells and CTCM, but no responding cells) by at
least 3 standard deviations of the mean.
| |
RESULTS |
Induction of split chimerism by purified
Sca-1+Lin progenitor cells in
sublethally irradiated allogeneic mice.
To test the potential of cells within the hematopoietic progenitor cell
fraction to overcome host resistance, we attempted to purify (using
magnetic beads) Sca-1+Lin BM cells from
C57BL/6 donors, and we tested their capacity to induce chimerism in
fully allogeneic C3H/HeJ recipients exposed to sub-lethal 7 Gy TBI.
Figure 1 shows an analysis on a
fluorescence-activated cell sorter (FACS) of a typical purification of
Sca-1+Lin cells afforded by the MACS
double-step procedure. The average frequency of these cells in the
initial BM fraction that was applied to the MACS procedure (depleted of
red blood cells and neutrophils by Ficoll separation) was 2.7% ± 1.1% (range, 1.8% to 5.1%), and after the 2-step fractionation
procedure it was enriched to an average of 68.3% ± 9.8% (range,
53.5% to 84.4%).
View larger version (28K):
[in this window]
[in a new window]
| Fig 1.
Purification of Sca-1+Lin BM
cells. Cells in the different cell fractions obtained by MACS
purification (see Materials and Methods) were analyzed by FACS for the
presence of Sca-1+Lin+,
Sca-1+Lin, and
Sca-1Lin+ cells. Nonspecific staining was
determined by FITC- and PE-conjugated isotype control antibodies.
Percentage of each subpopulation is shown in the appropriate area of
the dot plot.
|
|
Determination of the different subpopulations within the
Lin+ phenotype (Table 1) showed
more than 1 log depletion of CD4, CD8, CD11b, and CD45/B220 cells from
the Sca-1+Lin cell fraction compared
with that found in the Sca-1+ cell fraction. Determination
of donor-type chimerism after infusion of different cell numbers from
the Sca-1+Lin cell fraction showed an
exponential dose-response curve similar to those previously described
for T-cell-depleted transplants.5 The pooled results of 8 different experiments illustrating the chimerism level induced in each
individual mouse exposed to 7 Gy TBI clearly show that substantial
chimerism (>20%) was initially noticed upon infusion of 3 × 105 Sca-1+Lin cells (9 of
20), whereas recipients of 4 × 103 to 2 × 105 Sca-1+Lin cells very
rarely exhibited low levels of donor-type chimerism (2 of 48)
(Fig 2). In contrast, when C3H/HeJ
recipients of C57BL/6 Sca-1+Lin BM cells
were exposed to 10 Gy lethal TBI, about 75-fold fewer cells (4 × 103 Sca-1+Lin) were required
for the induction of over 50% donor-type chimerism in 10 of 11 recipients (Fig 2). It seems that a large proportion of the cells,
required to establish chimerism in the sublethal model, are involved in
overcoming the larger number of host immune cells remaining after 7 Gy
TBI, compared with the miniscule numbers surviving 10 Gy TBI.
Considering that, in C3H/HeJ recipients of C57BL/6 BM, resistance is
largely mediated by T-cell-mediated responses while NK-cell-mediated
resistance is minimal,29 it should be noted that spleens of
C3H/HeJ mice, 1 week after exposure to 7 Gy TBI, contain about 1 log
more T cells compared to mice treated with 10 Gy TBI (unpublished
results, January 1999). Indeed, FACS analysis of the chimeric mice
obtained after exposure to sublethal TBI showed a substantial number of
host-type T cells coexisting with donor-type T cells, as well as with
non-T cells (Fig 3). No GVHD, as
defined by measurement of body weight and the general appearance
of the mice, was observed in recipients of
Sca1+Lin cells, in agreement with
previous studies, defining the threshold for GVHD in lethally
irradiated mice30 by more than an order of magnitude above
the numbers that might have been infused in the present study
(ie, <1 × 103 T cell/mouse). Furthermore,
considering that antihost donor alloreactivity generally manifests
itself by eradication of host-type blood cells, our finding of
durable long-term substantial mixed chimerism strongly suggests that if
there is any T-cell contamination in the infused Sca1+Lin cell fraction (ie, below
detection of our FACS analysis; Table 1), it is not associated with any
form of clinical or subclinical GVHD.
View larger version (16K):
[in this window]
[in a new window]
| Fig 2.
Induction of donor type chimerism after transplantation
of C57BL/6 Sca-1+Lin cells into lethally
(10 Gy TBI) or sublethally (7 Gy TBI) irradiated C3H/HeJ recipients.
The number of Sca-1+Lin cells infused was
calculated based on FACS analysis.
|
|
View larger version (36K):
[in this window]
[in a new window]
| Fig 3.
Split chimerism after transplantation of C57BL/6
Sca-1+Lin cells (H2b) into
sublethally irradiated (7 Gy) C3H/HeJ recipients (H2k).
Percentages of donor and host T cells (determined by gating of CD3
stained cells) and non-T cells are shown in the appropriate FACS dot
plots. Peripheral blood chimerism was determined 30 days posttransplant
by cytofluorimetry (see Materials and Methods).
|
|
Limit dilution analysis of CTL-p in the spleen of chimeric mice showed
no detectable CTL-p against donor type cells (C57BL/6) while the
frequency of CTL-p against a third party (BALB/c) was not significantly different from that found in irradiated mice that
were not transplanted with Sca-1+Lin
cells (Fig 4). To test the capacity of
Sca-1+Lin cells to affect postthymic T
cells that survive the sublethal irradiation and that present a barrier
to engraftment during the immediate period posttransplant, we chose to
evaluate the level of chimerism at 30 days after transplantation.
However, we also tested donor-type chimerism 7 months posttransplant,
and it was found to be similar to the level found at the earlier time
point (data not shown).
View larger version (15K):
[in this window]
[in a new window]
| Fig 4.
Anti-donor CTL-p in allogeneic chimera (C57BL/6
 C3J/HeJ) generated by transplantation of C57BL/6
Sca-1+Lin cells. Limiting dilution
analysis of CTL-P was performed against donor (A) or third-party (B)
splenocytes. ( ) Irradiated mice transplanted with
Sca-1+Lin cells. ( )
Irradiated mice not receiving a transplant.
|
|
Therefore, it seems that cells within the purified
Sca-1+Lin cell fraction, or their
immediate progeny, can effectively tolerize the marked number of host
CTL-p that survive sublethal irradiation. Furthermore, when the
established chimera were challenged by skin grafts from donor or
third-party origin, only the former were accepted (7 of 7) while the
third-party skins were uniformly rejected (7 of 7). Thus, as in
radiation chimera following conditioning with lethal TBI, the tolerance
induced by the Sca-1+Lin cells in
sublethally irradiated recipients is not limited to hematopoietic antigens.
Enrichment for chimerism-inducing cells is associated both with
positive selection of Sca-1+ cells and with the subsequent
negative selection for Sca-1+ Lin cells.
Although the purification procedure afforded over 25-fold enrichment of
Sca-1+Lin cells, the average purity of
the Sca-1+Lin BM cell fraction achieved
by this large scale approach was limited to about 68%. Thus, it could
be argued that the tolerizing effect exhibited by the infusion of
Sca-1+Lin cells might be mediated in
part by other contaminating cells. Therefore, after each fractionation
step we evaluated both the enrichment factor of the selected phenotype
and the level of chimerism associated with this enrichment. In 2 experiments, no chimeric mice were found upon infusion of 1 × 106 unseparated cells, while following the first step of
positive selection of Sca-1+ cells, infusion of 1 × 106 cells of the Sca-1+ fraction led to
substantial donor type chimerism (>20%) in 10 of 14 recipients
(Fig 5). The second step of negative
selection led to further enhancement of engraftment (9 of 9) compared
with that observed in the Sca-1+ cell fraction. While the
latter enhancement is not statistically significant, there is clearly
no reduction of chimerism induction associated with the more than 1 log
depletion of Lin+ cells (Table 1), despite the exponential
nature of our chimerism induction curves (Fig 2). It seems that,
regardless of other cell phenotypes that might possess tolerizing or
facilitating activity, the enrichment of
Sca-1+Lin cells, associated with
depletion of different cell subpopulations sharing the
Sca-1+Lin+ phenotype, is also associated with
the capacity to achieve engraftment in the face of marked host immunity
remaining after the sublethal conditioning. Because several previous
studies have shown that different cell subpopulations bearing CD8 are
capable of enhancing BM allografts, we examined the presence of such
cells in the different cell fractions. The FACS data of a typical
experiment are illustrated in Fig 6, and
the level of each cell phenotype (based on the FACS analysis), found in
2 independent experiments, is shown in Table 1. The percentage of
CD8+ cells in the unseparated fraction was 0.6% to 0.7%.
After purification for Sca-1+ cells, the level of
CD8+ cells was enriched to 2.7% and 3.4%, respectively.
After depletion of Lin+ cells it was reduced to <0.1%
and <0.2%, respectively, in the Sca-1+Lin cell fraction. Thus, although
the enhancement of donor-type chimera in recipients of
Sca-1+ cells could be mediated by either
Sca-1+CD8+ or
Sca-1+Lin cells, both of which are
enriched in this fraction, the high level of chimerism found after the
second fractionation step, which is associated with at least a 20-fold
depletion of CD8+ cells along with a 12-fold enrichment of
Sca-1+Lin cells, strongly suggests that
a CD8-negative cell in the Sca-1+Lin
cell fraction is responsible for the ability of
Sca-1+Lin cells to induce marked
donor-type chimerism in face of the substantial host resistance
remaining in sublethally irradiated recipients.
View larger version (12K):
[in this window]
[in a new window]
| Fig 5.
Donor type chimerism after transplantation of different
BM cell fractions into allogeneic recipients exposed to sublethal (7 Gy) TBI: effect of positive selection for Sca-1+ cells
and subsequent depletion of Lin+ cells. The figure shows
peripheral blood chimerism at 30 days after transplantation of 1 × 106 cells from each cell fraction.
|
|
View larger version (41K):
[in this window]
[in a new window]
| Fig 6.
Depletion by MACS of CD8+,
CD4+, CD11b+, and CD45/B220+
cells after removal of Lin+ cells from the
Sca-1+ BM cell fraction. Levels of CD8+,
CD4+, CD11b+, and CD45/B220+
cells in unseparated, Sca-1+, and
Sca-1+Lin BM cell fractions, obtained by
MACS, were analyzed by double staining with antibodies directed against
Sca-1 and the above lineage markers. Nonspecific staining was
determined by FITC- and PE-conjugated isotype control antibodies.
|
|
Donor NK cells with antihost alloreactivity that might contaminate the
Sca-1+Lin cell fraction or emerge upon
engraftment and differentiation of the
Sca-1+Lin cells could also
contribute in part to the tolerance observed, by eradicating residual
host peripheral T cells. However, addition of anti-NK antibody to the
Lin+ depletion step did not abrogate the capacity of
Sca-1+Lin cells to engraft, nor did the
use of Sca-1+Lin cells from
NK-deficient C57BL/6-Beige donors (data not shown).
Specific reduction of antidonor CTL-p by
Sca-1+Lin cells.
To evaluate whether the Sca-1+Lin cell
fraction is enriched for cells possessing veto activity, we analyzed
different cell fractions for their capacity to specifically reduce
antidonor CTL-p in comparison with anti-third-party CTL-p. Mixed
lymphocyte reaction (MLR) was set up using spleen cells from C3H/HeJ
mice as responder cells and spleen cells from C57BL/6 or BALB/c
(third-party) mice as stimulator cells. To evaluate the veto activity
of C57BL/6 Sca-1+Lin cells compared with
Sca-1 cells, these cells were added to the MLR at a
veto-to-responder cell ratio of 0.5:1, and the CTL-p levels were
determined by limiting dilution analysis. As can be seen in
Table 2, addition of
Sca-1+Lin cells completely abrogated
CTL-p directed against their H-2 antigens (C57BL/6) while substantial
cytotoxic activity could be monitored when no cells were added, or upon
addition of Sca-1 cells. In contrast, when the same
Sca-1+Lin cells were added to MLR
against third-party (BALB/c) stimulators, no reduction of CTL activity
was found when compared with the level determined in the absence of
added cells. Thus, selection of
Sca-1+Lin cells is associated with
enrichment of veto activity.
Non-alloreactive (host × donor)F1 T cells synergize
with Sca-1+Lin BM cells of donor origin
in the induction of donor-type chimerism.
Although we are encouraged by the ability of
Sca-1+Lin cells to overcome the host T
cells remaining after sublethal TBI, it is clear that the number of
cells required is very high and that it may be quite difficult to
collect such large numbers of cells from human donors. For example, in
the experiments described above, transplantation into sublethally
irradiated recipients required about 75-fold more
Sca-1+Lin cells compared with the number
engrafting lethally irradiated mice. To reduce the effective number of
Sca-1+Lin cells needed for overcoming
resistance to engraftment, we were interested in studying the potential
role of other facilitating or veto cells. Therefore, we attempted to
test whether the addition of non-alloreactive (donor × host)F1 T cells, previously shown23 to exhibit
marked enhancement of engraftment of T-cell-depleted transplants in
mismatched lethally irradiated recipients, could also help to reduce
the minimal number of progenitor cells required for chimerism induction
in the sublethal model. As can be seen in
Fig 7, in 5 experiments
monitoring the engraftment of 1 × 107 infused
F1 T cells (H2b+H2k+) in
sublethally irradiated C3H/HeJ (H2k) recipients by their
unique H2b+H2k+ phenotype, we found that only
16.6% of the mice were engrafted, suggesting that infusion of this
number of F1 T cells alone could overcome resistance only
in a relatively small fraction of the recipients. However, engraftment
of F1 T cells was markedly increased to 65% by adding 2.0 × 105 purified
Sca-1+Lin cells. Likewise, engraftment
of a suboptimal number of 2.0 × 105 purified
(Sca-1+Lin) cells, monitored by the
presence of donor-type cells (stained by anti-H2b and not
by anti-H2k), was enhanced, by adding 1 × 107 F1 T cells, from 12.9% of the recipients
to 74.0%.
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
Donor type chimerism after transplantation of
Sca-1+Lin cells: the synergistic effect of
non-alloreactive T cells and purified
Sca-1+Lin cells. Different groups of
sublethally (7 Gy) irradiated C3H/HeJ recipients (H2k+)
were transplanted with 2 × 105 (C57BL/6)
Sca-1+Lin cells (H2b+), 10 × 106 (C3H/HeJ × C57BL/6)F1 T cells
(H2k+H2b+), or both. Peripheral blood
chimerism was analyzed 30 days posttransplant by flow cytometry,
monitoring the engraftment of Sca-1+Lin
cells (C57BL/6) (H2b+) (A) and (C3H/HeJ × C57BL/6)F1 T
cells (H2k+H2b+) (B). Data were pooled from
5 independent experiments.
|
|
The resulting chimeric mice exhibited stable split chimerism in which
significant levels of host (H2k+H2b) and
donor-type T cells (H2b+H2k) as well as
F1 T cells (H2b+H2k+) coexisted
with host and donor non-T cells (Fig 8).
View larger version (42K):
[in this window]
[in a new window]
| Fig 8.
A typical profile of split chimerism, exhibited by
chimeric mice transplanted with Sca-1+Lin
cells (2 × 105, H2b+), (C3H/HeJ × C57BL/6)F1 T cells (H2k+ + H2b+, 10 × 106), or both. Peripheral blood chimerism was analyzed 30 days posttransplant by flow cytometry, using antibodies directed
against host (H2k) and donor (H2b) MHC
antigens.
|
|
| |
DISCUSSION |
Our results demonstrate that cells within the BM
Sca-1+Lin subpopulation, previously
shown to comprise the pluripotential hematopoietic stem cells as well
as more differentiated progenitors, possess a marked capacity to
overcome the substantial host resistance found in recipients
conditioned by sublethal 7 Gy TBI. Considering that it is virtually
impossible to achieve homogeneous purity by any methodology of BM cell
fractionation, we resorted in our analysis to evaluation of specific
activities before and after each of the purification steps. We found
that the initial step of positive selection of Sca-1+ cells
contributes significantly to the enhancement of donor-type chimerism
and that over 1 log depletion of Lin+ cells from the
Sca-1+ cell fraction does not retract from the engraftment
potency of the resulting Sca-1+Lin cell
fraction but, rather, further enhances donor-type chimerism.
In particular, our demonstration that the chimerism-inducing activity
is enhanced upon purification of
Sca-1+Lin cells from the
Sca-1+ cell fraction, while the frequency of
CD8+ cells is reduced by about 20-fold, suggests that the
ability of Sca-1+Lin cells to overcome
the marked host immunity is probably mediated by a non-CD8-mediated
mechanism. Likewise, the use of NK-deficient "beige" donors or
NK-depleted Sca-1+Lin cells rule out the
possibility that the latter cells are critical for the effect exerted
by cells in the Sca-1+Lin cell fraction.
It could be argued that chimerism induction associated with
purification of Sca-1+Lin cells might
involve not only the capacity to overcome the host immune system, but
it may also be mediated by their capacity to compete with host
hematopoietic stem cells.31,32 This inherent duality of
Sca-1+Lin cells was addressed by our
demonstration that Sca-1+Lin cells
facilitate not only their own engraftment, which could be reflecting,
in part, donor-versus-host stem cell competition, but also the
engraftment of (donor × host)F1 T cells, as well as
acceptance of donor-type but not third-party skin. Thus, we were able
to separate in vivo the potent capacity to overcome host immunity
associated with the purified Sca-1+Lin
cells from their hematopoietic repopulating capacity, which might also
contribute to donor-type chimerism induction. We have shown recently
that escalation of human CD34 progenitor cell transplants enables
overcoming major HLA barriers in the treatment of heavily conditioned
leukemia patients.18-20 The capacity of the "mega
dose" transplants to neutralize residual host antidonor CTL-p was
attributed in part to the veto activity shown in the hematopoietic
progenitor CD34 cell compartment.21,22
Our present study, finding great similarity between the
Sca-1+Lin and the human CD34 cell
fraction, suggests that further insight into the mechanism of tolerance
induction by hematopoietic progenitor cells might be facilitated by
using different natural or genetically engineered mutant strains of mice.
Our demonstration that resistance to fully allogeneic stem cell
transplants found in sublethally irradiated recipients can be overcome
by escalation of cell dose is in accordance with the recent study of
Uchida et al,33 which showed that lethally irradiated allogeneic recipients transplanted with highly purified
c-Kit+Thy-1.1lo-Lin/loSca-1+
stem cells require about 10-fold more cells compared with congenic recipients. However, the comparison between the allogeneic and the
congenic groups in that study was made using animals pretreated by a
different TBI dose. Moreover, there are no data regarding recipients
treated with sublethal TBI. Thus, further studies are required to
define whether the
c-Kit+Thy-1.1lo-Lin/loSca-1+
cells within the Sac-1+Line cell
fraction will exhibit a similar capacity to overcome BM allograft
rejection to that exhibited in the present study by the
Sca-1+Line cells in the
sublethal model.
The characterization of new cell phenotypes that are capable of
enhancing acceptance of T-cell-depleted BM allografts is of great
relevance to the continuing efforts to achieve HLA disparate hematopoietic engraftment in humans. Our present results in the mouse
model, although demonstrating that major transplantation barriers can
be overcome by megadoses of BM progenitors, also suggest that it might
be difficult to harvest sufficient cells in humans. We found that the
minimal number of Sca-1+Lin cells
required to achieve donor type chimerism in 7 Gy TBI treated mice was
about 75-fold higher than that needed in lethally irradiated (10 Gy
TBI) recipients.
One approach to overcome this quantitative problem might be afforded if
it were possible to expand the veto cells within the CD34+
progenitor cell fraction. Thus, while it is still very difficult to
expand ex vivo the most primitive pluripotential hematopoietic stem
cells, it may be possible to expand in vitro the CD34+
cells possessing veto activity and use them together with a small number of pluripotential cells for transplantation. Further studies to
elucidate the cell subpopulations that are most relevant for the
induction of tolerance, in the human CD34 or in the mouse Sca-1+Lin subpopulation, might help to
find simple means to expand these valuable cells in vitro, and
could help develop strategies for the elucidation of the mechanism by
which their effect is mediated.
An alternative approach to achieving engraftment in the face of marked
residual host immunity is based on the use of CD34 cells, together with
other veto cells or facilitating cells. One example for such cells is
shown in the present study by using (host × donor)F1
non-alloreactive T cells, previously shown to synergize
with suboptimal doses of T-cell-depleted BM allografts in the lethal
mouse model.30 In humans, non-alloreactive T cells can be
generated by purging interleukin-2 receptor (CD25) MLR reactive T
cells34 or by anergy induction upon incubation with CTLA-4.35,36
| |
FOOTNOTES |
Submitted August 24, 1998; accepted June 22, 1999.
Supported in part by a grant from the Israel Academy of Science, the
Leukemia Society of America, and the Rich 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. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yair Reisner, Department of Immunology, The
Weizmann Institute of Science, Rehovot 76100, Israel.
| |
REFERENCES |
1.
Qin S, Cobbold SP, Pope H, Elliott J, Kioussis D, Davies J, Waldmann H:
"Infectious" transplantation tolerance.
Science
259:974, 1993[Abstract]
2.
Wood K, Sachs DH:
Chimerism and transplantation tolerance: Cause and effect.
Immunol Today
17:584, 1996[Medline]
[Order article via Infotrieve]
3.
Greenstein JL, Sachs DH:
The use of tolerance for transplantation across xenogeneic barriers.
Nat Biotechnol
15:235, 1997[Medline]
[Order article via Infotrieve]
4.
Reisner Y:
Chimerism, hematopoietic, in
Roitt IM,
Delves PJ
(eds):
Encyclopedia of Immunology. London, UK, Academic, 1992, p 332
5.
Bachar-Lustig E, Rachamim N, Li HW, Lan F, Reisner Y:
Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice.
Nat Med
1:1268, 1995[Medline]
[Order article via Infotrieve]
6.
Sykes M, Szot GL, Swenson KA, Pearson DA:
Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning.
Nat Med
3:783, 1997[Medline]
[Order article via Infotrieve]
7.
Cobbold SP, Martin G, Qin S, Waldmann H:
Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance.
Nature
323:164, 1986[Medline]
[Order article via Infotrieve]
8.
Strober S, Palathumpat V, Schwadron R, Hertel-Wulff B:
Cloned natural suppressor cells prevent lethal graft-vs-host disease.
J Immunol
138:699, 1987[Abstract]
9.
Kikuya S, Inaba M, Ogata H, Yasumizu R, Inaba K:
Wheat germ agglutinin-positive cells in a stem cell-enriched fraction of mouse bone marrow have potent natural suppressor activity.
Proc Natl Acad Sci USA
85:4824, 1988[Abstract/Free Full Text]
10.
Tscherning T, Claesson M:
Veto-like down regulation of T helper cell reactivity in vivo by injection of semi-allogeneic spleen cells.
Immunol Letters
29:223, 1991[Medline]
[Order article via Infotrieve]
11.
Kiyoshi H, Hiruma K, Nakamura H, Henkart P, Gress R:
Clonal deletion of postthymic T cells: Veto cells kill precursor cytotoxic T lymphocytes.
J Exp Med
175:863, 1992[Abstract/Free Full Text]
12.
Kaufman CL, Colson YL, Wren SM, Watkins S, Simmons RL, Ildstad ST:
Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells.
Blood
84:2436, 1994[Abstract/Free Full Text]
13.
Pierce GE, Watts LM:
Do donor cells function as veto cells in the induction and maintenance of tolerance across an MHC disparity in mixed lymphoid radiation chimeras?
Transplantation
55:882, 1993[Medline]
[Order article via Infotrieve]
14.
Pierce GE, Watts LM:
Thy 1+ donor cells function as veto cells in the maintenance of tolerance across a major histocompatibility complex disparity in mixed-lymphoid radiation chimeras.
Transplant Proc
25:331, 1993[Medline]
[Order article via Infotrieve]
15.
Sambhara SR, Miller RG:
Programmed cell death of T cells signaled by the T cell receptor and the alpha 3 domain of class I MHC.
Science
252:1424, 1991[Abstract/Free Full Text]
16.
Thomas JM, Carver FM, Cunningham PR, Olson LC, Thomas FT:
Kidney allograft tolerance in primates without chronic immunosuppression The role of veto cells.
Transplantation
51:198, 1991[Medline]
[Order article via Infotrieve]
17.
Qi Y, Berg R, Singleton MA, Debrick JE, Staerz DU:
Hybrid antibody mediated veto of cytotoxic T lymphocyte responses.
J Exp Med
183:1973, 1996[Abstract/Free Full Text]
18.
Aversa F, Tabilio A, Terenzi A, Velardi A, Falzetti F, Giannoni C, Iacucci R, Zei T, Martelli MP, Gambelunghe C, Rossetti M, Caputo P, Latini P, Aristei C, Raymondi C, Reisner Y, Martelli MF:
Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum.
Blood
84:3948, 1994[Abstract/Free Full Text]
19.
Reisner Y, Martelli MF:
Bone marrow transplantation across HLA barriers by increasing the number of transplanted cells.
Immunol Today
16:437, 1995[Medline]
[Order article via Infotrieve]
20.
Aversa F, Tabilio A, Velardi A, Cunningham I, Terenzi A, Falzetti F, Ruggeri L, Barbabietola G, Aristei C, Latini P, Reisner Y, Martelli MF:
Transplantation of high-risk acute leukemia with T-cell-depleted stem cells from related donor with one fully mismatched HLA haplotype.
N Engl J Med
339:1186, 1998[Abstract/Free Full Text]
21.
Rachamim N, Gan Y, Segal H, Marcus H, Berrrebi A, Krauthgamer R, Martelli MF, Reisner Y:
Potential role of CD34 stem cells in tolerance induction.
Transplant Proc
29:1935, 1997[Medline]
[Order article via Infotrieve]
22.
Rachamim N, Gan J, Segall H, Krauthgamer R, Marcus H, Berrrebi A, Martelli M, Reisner Y:
Tolerance induction by "megadose" hematopoietic transplants: Donor-type human CD34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture.
Transplantation
65:1386, 1998[Medline]
[Order article via Infotrieve]
23.
Lapidot T, Faktorowich Y, Lubin I, Reisner Y:
Enhancement of T cell-depleted bone marrow allografts in the absence of graft-versus-host disease is mediated by CD8+CD4 and not by CD8CD4+ thymocytes.
Blood
80:2406, 1992[Abstract/Free Full Text]
24.
Levite M, Meshorer A, Reisner Y:
A rapid method for obtaining murine bone marrow cells in high yield.
Bone Marrow Transpl
8:1, 1991[Medline]
[Order article via Infotrieve]
25.
Spangrude GJ, Scollay R:
A simplified method for enrichment of mouse hematopoietic stem cells.
Exp Hematol
18:920, 1990[Medline]
[Order article via Infotrieve]
26.
Reisner Y, Linker-Israel M, Sharon N:
Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin.
Cell Immunol
25:129, 1976[Medline]
[Order article via Infotrieve]
27.
Baharov E, Lider O, Margalit M, Cohen IR:
A modified technique for experimental skin grafting.
J Immunol Methods
90:143, 1986[Medline]
[Order article via Infotrieve]
28.
Ryser JE, MacDonald HR:
Limiting dilution analysis of alloantigen-reactive T lymphocytes. I. Comparison of precursor frequencies for proliferative and cytolytic responses.
J Immunol
122:1691, 1979[Abstract/Free Full Text]
29.
Sentman CL, Kumar V, Koo G, Bennett M:
Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts.
J Immunol
142:1847, 1989[Abstract]
30.
Lapidot T, Lubin I, Terenzi A, Faktorowich Y, Erlich P, Reisner Y:
Enhancement of bone marrow allografts from nude mice into mismatched recipients by T cells void of graft-versus-host activity.
Proc Natl Acad Sci USA
87:4595, 1990[Abstract/Free Full Text]
31.
Lapidot T, Terenzi A, Singer TS, Salomon O, Reisner Y:
Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts.
Blood
73:2025, 1989[Abstract/Free Full Text]
32.
Stewart FM, Zhong S, Wuu J, Hsieh C, Nilsson SK, Quesenberry PJ:
Lymphohematopoietic engraftment in minimally myeloablated hosts.
Blood
86:924, 1995[Abstract/Free Full Text]
33.
Uchida N, Tsukamoto A, He D, Friera AM, Scollay R, Weissman IL:
High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts.
J Clin Invest
101:961, 1998[Medline]
[Order article via Infotrieve]
34.
Cavazzana Calvo M, Stephan JL, Sarnacki S, Chevret S, Fromont C, De Coene C, Le Deist F, Guy Grand D, Fischer A:
Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination.
Blood
83:288, 1994[Abstract/Free Full Text]
35.
Boussiotis VA, Gribben JG, Freeman GJ, Nadler LM:
Blockade of the CD28 co-stimulatory pathway: A means to induce tolerance.
Curr Opin Immunol
6:797, 1994[Medline]
[Order article via Infotrieve]
36.
Gribben JG, Guinan EC, Boussiotis VA, Ke XY, Linsley L, Sieff C, Gray GS, Freeman GJ, Nadler LM:
Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: A method to ameliorate graft-versus-host disease and extend the donor pool.
Blood
87:4887, 1996[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. Mariotti, J. Foley, U. Jung, T. Borenstein, N. Kantardzic, S. Han, J. T. Hanson, E. Wong, N. Buxhoeveden, J. B. Trepel, et al.
Ex Vivo Rapamycin Generates Apoptosis-Resistant Donor Th2 Cells That Persist In Vivo and Prevent Hemopoietic Stem Cell Graft Rejection
J. Immunol.,
January 1, 2008;
180(1):
89 - 105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Pearl-Yafe, E. S. Yolcu, J. Stein, O. Kaplan, I. Yaniv, H. Shirwan, and N. Askenasy
Fas Ligand Enhances Hematopoietic Cell Engraftment Through Abrogation of Alloimmune Responses and Nonimmunogenic Interactions
Stem Cells,
June 1, 2007;
25(6):
1448 - 1455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. D. Arditti, S. Aviner, B. Dekel, R. Krauthgamer, J. Gan, A. Nagler, A. Tabilio, M. Martelli, A. Berrebi, and Y. Reisner
Eradication of B-CLL by autologous and allogeneic host nonreactive anti-third-party CTLs
Blood,
April 15, 2005;
105(8):
3365 - 3371.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Gur, R. Krauthgamer, E. Bachar-Lustig, H. Katchman, R. Arbel-Goren, A. Berrebi, T. Klein, A. Nagler, A. Tabilio, M. F. Martelli, et al.
Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-{alpha}
Blood,
March 15, 2005;
105(6):
2585 - 2593.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Reich-Zeliger, E. Bachar-Lustig, J. Gan, and Y. Reisner
Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. I. Relative Reactivity of Different Veto Cells
J. Immunol.,
December 1, 2004;
173(11):
6654 - 6659.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Reich-Zeliger, J. Gan, E. Bachar-Lustig, and Y. Reisner
Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. II. Deletion of Effector Cells by Fas-Fas Ligand Apoptosis
J. Immunol.,
December 1, 2004;
173(11):
6660 - 6666.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Bachar-Lustig, S. Reich-Zeliger, and Y. Reisner
Anti-third-party veto CTLs overcome rejection of hematopoietic allografts: synergism with rapamycin and BM cell dose
Blood,
September 15, 2003;
102(6):
1943 - 1950.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Gur, R. Krauthgamer, A. Berrebi, T. Klein, A. Nagler, A. Tabilio, M. F. Martelli, and Y. Reisner
Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34+ cells
Blood,
May 13, 2002;
99(11):
4174 - 4181.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. E. Georges, R. Storb, B. Bruno, S. J. Brodie, J. D. Thompson, A. G. Taranova, J. M. Zaucha, M.-T. Little, E. Zellmer, P. F. Moore, et al.
Engraftment of DLA-haploidentical marrow with ex vivo expanded, retrovirally transduced cytotoxic T lymphocytes
Blood,
December 1, 2001;
98(12):
3447 - 3455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION