Tolerance Induction by Veto CTLs in the
TCR Transgenic 2C Mouse Model. I. Relative
Reactivity of Different Veto Cells
This information is current as
of June 17, 2017.
Shlomit Reich-Zeliger, Esther Bachar-Lustig, Judith Gan and
Yair Reisner
J Immunol 2004; 173:6654-6659; ;
doi: 10.4049/jimmunol.173.11.6654
http://www.jimmunol.org/content/173/11/6654
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References
The Journal of Immunology
Tolerance Induction by Veto CTLs in the TCR Transgenic 2C
Mouse Model. I. Relative Reactivity of Different Veto Cells1
Shlomit Reich-Zeliger, Esther Bachar-Lustig, Judith Gan, and Yair Reisner2
B
one marrow transplantation after supralethal radiochemotherapy is associated with dangerous infections due to
the slow immune reconstitution during the first year posttransplant (1–3). Thus, the use of reduced intensity conditioning,
associated with less severe immune ablation, could have a remarkable potential in the treatment of a variety of nonmalignant diseases or for the induction of mixed chimerism as a prelude for cell
therapy in cancer or organ transplantation (4 –7). However, the
marked level of host hemopoietic and immune cells surviving mild
preparatory regimens represents a difficult barrier for the engraftment of donor cells.
In patients with advanced hematological malignancies who cannot withstand myeloablative conditioning because of age and/or
performance status, recent attempts were made to develop low
toxicity conditioning protocols in conjunction with HLA-matched
transplants. Potent post-transplant immunosuppression and the
presence of large numbers of alloreactive T cells in the graft enabled a high rate of engraftment. However, graft-vs-host disease
(GvHD),3 particularly chronic GvHD, remains a major obstacle
(7). Although in high risk leukemia, such transplant-related mortality is acceptable, it would be totally intolerable if applied to
patients with long life expectancy. Thus, the use of purified allogeneic stem cells, which do not pose any risk for GvHD and which
can continuously present donor type Ags in the host thymus,
thereby inducing durable tolerance to donor cells or tissues, represents one of the most desirable goals in transplantation biology.
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Received for publication March 25, 2004. Accepted for publication September
23, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grant CA49369,
Therapy of CML; and grants from E. Drake; and the Gabriella Rich Center for Transplantation Biology Research.
2
Address correspondence and reprint requests to Dr. Yair Reisner, Department of
Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address:
[email protected]
3
Abbreviations used in this paper: GvHD, graft-vs-host disease; Tg, transgenic.
Copyright © 2004 by The American Association of Immunologists, Inc.
One approach to address this challenge is based on the use of
donor veto cells depleted of host reactive T cells.
Veto activity was defined in 1980 by Miller as the capacity to
specifically suppress cytotoxic T cell precursors directed against
Ags of the veto cells themselves, but not against third-party Ags
(8 –11). 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 (12–
17). The specificity of the veto effect mediated by CTL clones was
shown by several studies to be unrelated to their TCR specificity
(18 –20). The suppression of effector CTL-precursor directed
against the veto cells is both Ag-specific and MHC-restricted, resulting from the unidirectional recognition of the veto cell by the
responding CTLs, but not vice versa (18). Furthermore, it has been
shown that this suppression is mediated by apoptosis (18, 21).
Although CD8⫹ CTLs are endowed with very potent veto activity, their use in allogeneic stem cell transplantation is limited
due to their marked graft-vs-host reactivity. To address this problem we developed a new approach for the generation of host-nonreactive CTLs based on stimulation of donor CD8⫹ T cells against
third-party stimulators under IL-2 deprivation. This approach is
based on the observation that only those CTLs activated are capable of surviving IL-2 starvation in the primary culture. Other
studies used T cytotoxic 2 CTLs, which were shown to be associated with a reduced GVH reactivity (22), or NK cells (23), which
are not associated with any risk for GVHD. One obstacle in the
study of basic questions related to the mechanism of action of veto
cells is presented by the assay for these cells. Due to the low
frequency of the effector T cells against which the veto cell inhibitory activity is directed, the fate of the effector cells was traditionally followed indirectly by functional limiting dilution assays,
which are cumbersome and depend on numerous parameters.
However, the availability of new TCR transgenic (Tg) strains of
mice, in which the transgene is directed against H-2 determinants,
as well as the development of tetramer technology have made it
possible to directly monitor in such MLR cultures the fate of the
effector T cells by FACS analysis. Thus, the 2C Tg mouse model,
the T cells of which bear a transgene directed against H-2d, was
used to document the veto reactivity of activated bone marrow
0022-1767/04/$02.00
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Several bone marrow cells and lymphocyte subpopulations, known as veto cells, were shown to induce transplantation tolerance
across major histocompatibility Ags. Due to the low frequency of the effector T cells against which the veto cells inhibitory activity
is aimed, the fate of the effector cells was traditionally followed indirectly by functional limiting dilution assays, which are
cumbersome and depend on numerous parameters. In the present study the fate of the effector T cells was monitored directly by
FACS, using TCR transgenic mouse CD8ⴙ T cells in which the transgene is directed against H-2d (the 2C model). This assay is
validated by demonstrating the potency, selectivity, radiation sensitivity, and contact dependency of anti-third-party CTLs previously demonstrated by the limiting dilution assay. In contrast to veto CTLs, nonactivated CD8ⴙ T cells lack veto activity.
Comparison by FACS in the 2C model revealed a hierarchy of veto cells, in the order of veto CTLs activated NK cells, activated
CD4ⴙ T cells, and activated B cells. The latter cells as well as nonactivated CD4ⴙ or NK cells were shown to be completely devoid
of veto activity. The Journal of Immunology, 2004, 173: 6654 – 6659.
The Journal of Immunology
6655
cells (21, 23, 24). In the present study we used this Tg mouse
model to study the relative veto reactivities of various veto cells,
including host nonreactive CD8⫹ CTLs, activated CD4⫹ T cells,
B cells, and NK cells.
Biotec). The purified cells were then cultured in the presence of LPS (20
␮g/106 cells; Difco, Detroit, MI). On day 4, the cultures were harvested,
fractionated on Ficoll-Paque Plus (Amersham Biosciences), and analyzed
by FACS for their veto activity at different cell ratios, as described in
Results.
Materials and Methods
Assay for veto activity in the 2C TCR Tg mouse model
Animals
The mice used were 6- to 12-wk-old females. BALB/c (H-2d), FVB (H-2q),
SJL (H-2s), and C57BL/6 (H-2b) were obtained from the Weizmann Institute Animal Center (Rehovot, Israel). DBA/2 (H-2d) and C3H/HeJ (H-2k)
mice were obtained from the Roscoe B. Jackson Memorial Laboratory (Bar
Harbor, ME). A breeding pair of Tg H-2b mice expressing the TCR from
the CTL clone 2C with specificity for H-2Ld was provided by J. NikolicZugic (Sloan-Kettering Institute, New York, NY). Progeny of these Tg
mice were bred at the Weizmann Institute Animal Breeding Center. All
mice were kept in small cages (five animals in each cage) and were fed
sterile food and acid water.
Preparation of host-nonreactive, donor anti-third-party CTLs
⫹
Preparation of host-nonreactive, anti-third-party CD4 cells
Splenocytes of BALB/c or FVB mice (nonspecific H-2 control) were harvested, and single cell suspensions were prepared. The cell suspensions
were treated with Tris-buffered ammonium chloride to remove RBC, and
the isolated mononuclear cells (2 ⫻ 106/ml) were stimulated with irradiated (20 Gy) C3H/HeJ or SJL (third-party stimulators) splenocytes (2 ⫻
106/ml). Responders and stimulators were cultured for 10 days in a complete tissue culture medium at 37°C in a 5% CO2/air incubator. Six days
after culture initiation, cells were fractionated on Ficoll, and the lymphoid
fraction was then subjected to positive selection of CD4⫹ cells using magnetically labeled anti-CD4 Abs and a MACS system (Miltenyi Biotec).
Human rIL-2 (40 U/ml; Eurocetus) was added every second day to the
MLR culture. On day 10, the MLR cultures were harvested, fractionated on
Ficoll-Paque Plus (Amersham Biosciences), analyzed by FACS for their
CD4 level, and tested for their veto activity at different cell ratios, as described in Results.
Cytofluorometric analysis
FACS analysis was performed using a modified FACScan (BD Biosciences, Mountain View, CA). Fluorescence data were collected using
3-decade logarithmic amplification on 25–50 ⫻ 103 viable cells, as determined by forward light scatter intensity. Cells were stained with anti-CD8␣
(Ly-2)-FITC, anti-CD8␣ (Ly-2)-CyChrome, anti-CD8␣ (Ly-2)-allophycocyanin, anti-CD3⑀-PE, anti-CD95 (Fas)-FITC (BD Pharmingen, San Diego, CA), and anti-CD4-Quantum Red (␴). Biotinylated 1B2 Abs (provided by J. Nikolic-Zugic, Sloan-Kettering Institute) were stained with
R-PE streptavidin (Jackson ImmunoResearch Laboratories, West
Grove, PA).
Detection of apoptotic cells
Annexin V-Cy5 was used to detect apoptotic cells. Cells were incubated in
annexin V binding buffer (26 –28) and supplemented with 5 ␮l of annexin
V-Cy5. The cells were incubated at room temperature for 5 min in the dark,
then washed in binding buffer. Positive cells were monitored by flow
cytometry.
Results
Veto activity of anti-third-party CTLs in the 2C TCR Tg
mouse model
Direct FACS analysis of veto activity. The TCR Tg anti-H-2d
CD8⫹ T cells of the 2C mouse can be identified by immunostaining with a clonotypic Ab directed against the transgene TCR.
Thus, it is possible to monitor by FACS the induction of apoptosis
of the effector cells by specific veto cells.
To adapt this model for the study of veto cells, anti-third-party
veto CTLs, which were previously characterized by functional inhibition of specific target killing (limiting dilution assay of the Cr
release assay), were initially used. In these experiments, anti-thirdparty CTLs generated from the BALB/c (H-2d) or FVB/N (H-2q)
Preparation of activated NK cells
Splenocytes of BALB/c mice or FVB mice were harvested, and single cell
suspensions were prepared. The cell suspensions were treated with Trisbuffered ammonium chloride to remove RBC, and the isolated mononuclear cells (2 ⫻ 106/ml) were subjected to positive selection of NK cells
using magnetically labeled anti-NK Abs (DEX-5) and a MACS system
(Miltenyi Biotec). Human rIL-2 (1500 U/ml; Eurocetus) was added to the
culture. On day 4, the cultures were harvested, fractionated on Ficoll-Paque
Plus (Amersham Biosciences), analyzed by FACS for their Dex-5 Ag level,
and tested for their veto activity at different cell ratios, as described in
Results.
Preparation of activated B cells
Splenocytes of BALB/c mice or FVB mice were harvested, and single cell
suspensions were prepared. The cell suspensions were treated with Trisbuffered ammonium chloride to remove RBC, and the isolated mononuclear cells (2 ⫻ 106/ml) were subjected to positive selection of B cells
using magnetically labeled anti-B-220 Abs and a MACS system (Miltenyi
FIGURE 1. Specific inhibition of responder CTL-precursor directed
against the H-2 of the veto cells. Splenocytes from 2C Tg mice (H-2b)
bearing transgene TCR specific against H-2d (1B2⫹) were stimulated in
MLR against BALB/c splenocytes (H-2d). Double-staining by FACS
shows the frequency of 1B2⫹ CD8 T cells in the absence (A) or the presence of anti-third-party CTLs of BALB/c origin (B) or CTLs of FVB origin
(C) 3 days after initiation of the MLR.
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Anti-third-party CTLs were prepared as described by Bachar-Lustig et al.
(25). Briefly, splenocytes of BALB/c or DBA/2 origin were cultured
against irradiated (20 Gy) FVB, C3H/HeJ. or SJL (third-party stimulators)
splenocytes. Responders (2 ⫻ 106/ml) and stimulators (2 ⫻ 106/ml) were
cultured for 6 days in RPMI 1640 complete tissue culture medium at 37°C
in a 5% CO2/air incubator. Six days after culture initiation, cells were
fractionated on Ficoll, and the lymphoid fraction was subjected to positive
selection of CD8⫹ cells using magnetically labeled anti-CD8 Abs and a
MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells (2 ⫻ 106/ml) were restimulated with irradiated (20 Gy) splenocytes from the original third-party donors (FVB, C3H/HeJ, or SJL; 2 ⫻
106/ml), and human rIL-2 (40 U/ml; Eurocetus, Milan, Italy) was added,
beginning that day, every second day to the MLR culture (days 6, 8, and
10). On day 10, the MLR cultures were harvested, fractionated on FicollPaque Plus (Amersham Biosciences, Uppsala, Sweden), analyzed by FACS
for CD8 level, and tested for veto activity at different cell ratios, as described in Results.
Spleen cells of 2C Tg H-2b mice, expressing the TCR-␣␤ with specificity
for H-2Ld mice (provided by J. Nikolic-Zugic, Sloan-Kettering Institute),
were collected as described above. The cells (2 ⫻ 106/ml) were then stimulated with irradiated (20 Gy) BALB/c splenocytes (2 ⫻ 106/ml) in the
presence or the absence of 20, 10, or 2% cells of specific (BALB/c origin)
and nonspecific (FVB origin) veto CTLs. Cultures were incubated for 72 h
in 24-well plates. The deletion of specific effector T cells was monitored by
cytofluorometric analysis, measuring the level of 2C Tg cells, specifically
stained by the 1B2 Ab, directed against the clonotypic anti-H-2Ld TCR.
6656
TOLERANCE INDUCTION BY VETO CTLs
Table I. Veto activity of anti-third party CTLsa
SJL (H-2s) Anti-C3H/Hej CTL (Nonspecific)
BALB/C (H-2d) Anti-C3H/Hej CTL (Specific)
% 1B2⫹CD8⫹ cells
% 1B2⫹CD8⫹ cells
Veto:Effector Ratio
Without
veto cells
With veto
cells
% Inhibition
t test
Without
veto cells
With veto
cells
% Inhibition
t test
0.0025
0.005
0.01
0.02
0.05
0.1
0.2
53
49
48
40
41
43
46
59
46
48
43
40
36
22
1
6
1
1
2
16
51
1
0.7
1
1
0.9
0.03
0.014
53
49
48
40
41
43
46
48
28
22
16
9
8
5
9
43
54
60
78
81
90
0.06
0.05
1 ⫻ 10⫺3
4 ⫻ 10⫺4
1 ⫻ 10⫺5
2 ⫻ 10⫺5
5 ⫻ 10⫺5
a
Results expressed as a percentage of inhibition are calculated as follows: % inhibition ⫽ 1 ⫺ (frequency of 1B2⫹CD8⫹
cells in the presence of veto cells)/(frequency 1B2⫹CD8⫹ cells in the absence of veto cells) ⫻ 100. The SD of replicate values
was consistently ⬍10% of the mean.
T cell activation is required for veto reactivity. To evaluate the
role of cell activation in veto activity, we compared CD8⫹ veto
CTLs of F1 origin to fresh CD8⫹ spleen T cells. As shown in Table
II, the latter cells exhibit only marginal veto activity. Likewise,
fresh CD8⫹ T cells purified from alloreactive mice (sharing the
H-2 determinants of the stimulator cells) did not exhibit veto activity, similar to the level exhibited in the control group by fresh
CD8⫹ T cells purified from alloreactive mice not sharing the H-2
determinants of the stimulator cells.
Role of cell contact and veto cell irradiation
As previously suggested by other indirect assays, the veto activity
of the CTLs is dependent on cell contact. Thus, upon separating
veto CTLs from effector cells in Transwell plates, the veto activity
was completely lost (Table II). Likewise, irradiation of the veto
cells abrogated their reactivity (Table II).
Relative potencies of different veto cells
Several lymphocyte subpopulations and bone marrow cells were
shown to exhibit veto activity (29 – 41). The establishment of a
quick and effective direct assay using 2C TCR Tg murine cells
enabled us to compare the relative potencies of different veto cells
using the same scale. Thus, the veto activity of anti-third-party
CTLs in the 2C model, reflected by expansion inhibition of
CD8⫹1B2⫹ effector cells, was compared with that exhibited by the
following lymphocyte subpopulations: 1) resting CD4⫹ T cells, 2)
resting CD8⫹ T cells, 3) resting NK cells, 4) activated CD4⫹ T
cells, 5) LPS-activated B cells, and 6) IL-2-activated NK cells. As
Table II. Veto activity of anti-third party CTLs of donor type and of F1 (host ⫻ donor) origin: the role of
activation, cell contact, and irradiation
BALB/c Splenocytes
(Specific)
SJL Splenocytes
(Nonspecific)
F1 (BALB/c ⫻ C57BL/6)
Splenocytes (Specific)
% Inhibitiona
Cells/Veto Cells Origin
Anti-third party CTLs (n ⫽ 20)
Fresh CD8 cells (n ⫽ 3)
Irradiated CTLs (n ⫽ 3)
CTLs in Transwell (n ⫽ 3)
A
B
78 ⫾ 9 91 ⫾ 9
18 ⫾ 8 20 ⫾ 8
4 ⫾ 3 7.5 ⫾ 6.5
1 ⫾ 1 7.5 ⫾ 1
A
B
A
B
20 ⫾ 12.3
2.5 ⫾ 2.5
4.5 ⫾ 3.5
1⫾1
15 ⫾ 13
6⫾6
5.5 ⫾ 2.5
75.6 ⫾ 8.1
18 ⫾ 11
86 ⫾ 11
20 ⫾ 5
a
Results are expressed as a percentage of inhibition, calculated as follows: % inhibition ⫽ 1 ⫺ (frequency of 1B2⫹CD8⫹
cells in the presence of veto cells)/(frequency 1B2⫹ CD8⫹ cells in the absence of veto cells) ⫻ 100. The inhibition was measured
at veto:effector cell ratio of 1:20 (A) or 1:10 (B).
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background were added to MLR cultures of 2C effectors stimulated against irradiated BALB/c splenocytes.
As shown in Fig. 1 (a representative experiment of 17 different
experiments), marked expansion of 2C cells was found when cells
were stimulated in the absence of veto CTLs. This expansion of 2C
effector cells, which were double-stained with anti-CD8 and the
clonotypic Ab 1B2, was markedly inhibited upon the addition of
anti-third-party CTLs from the BALB/c background. In contrast,
the addition of anti-third-party veto CTLs generated from FVB/N
splenocytes to the same culture at the same concentration only
slightly inhibited the expansion of CD8⫹1B2⫹ responder cells.
The specificity of the veto CTLs was further demonstrated by measuring the inhibition at different veto:effector cell ratios (Table I).
Thus, although marked reactivity was exhibited at a very low ratio
(43% inhibition at a 0.005 veto:effector ratio), only insignificant
inhibition was displayed by CTL of C3H background (H-2k, not
recognized by 2C effectors) even at a ratio of 0.05 (2% inhibition).
A more pronounced nonspecific inhibition was first detected at a
0.1 veto:effector cell ratio. The specificity revealed by these results
suggests that the host-nonreactive CTLs directed against thirdparty cells indeed exhibit veto activity and rules out potential elimination of effector cells by residual alloreactive clones, which
might still be present in the veto CTL preparation. This conclusion
is supported by veto experiments in which the anti-third-party
CTLs were generated from F1 mice, which lack any alloreactivity
against the recipients by virtue of their genetic background (progeny of breeding the veto cell strain with the effector cell strain). As
shown in Table II, the veto effect of F1 CTLs was similar to that
exhibited by the parent anti-third-party CTLs.
The Journal of Immunology
Table III. The relative veto activity of activated lymphocyte
subpopulations
6657
lead to any appreciable inhibition up to a veto:effector ratio of 0.2
( p ⫽ 0.005; Table III).
By this analysis, activated NK cells exhibited lower veto activity, attaining significance at a veto:effector ratio of 0.1 ( p ⫽ 0.004;
Table III). However, considering that at a 0.05 ratio the inhibition
was close to being significant ( p ⫽ 0.06), it could be that effective
inhibition might have been attained between a veto:effector ratio of
0.05 and 0.1, suggesting 10- to 20-fold reduction in veto activity
compared with veto CTLs.
Activated CD4 cells exhibit inhibition only at a veto:effector
ratio of 0.2, suggesting reduction of veto activity compared with
NK cells by 2- to 5-fold, whereas activated B cells are completely
devoid of veto activity.
Finally, all cell types tested failed to exhibit veto activity if not
activated before addition to the 2C culture (Fig. 2).
Discussion
shown in Table I, in which the veto activity of anti-third-party
CTLs was evaluated at different veto:effector cell ratios, and in
Table III, in which the inhibitory activity of the different activated
cells is shown, anti-third-party CTLs exhibit the most potent veto
activity. By this analysis we define the lowest veto:effector cell
ratio at which significant veto activity is exhibited compared with
the cell ratio at which inhibition is found with nonspecific test cells
(from the H2 background not recognized by the 2C effectors).
Thus, whereas a significant inhibition of 2C expansion was attained at a veto:effector cell ratio of 0.005 ( p ⫽ 0.05; Table I),
addition of anti-third-party CTLs of the H-2q background did not
FIGURE 2. The relative veto activities of different lymphocyte subpopulations. The inhibitory effects of CD8 cells (f), NK cells (_), CD4
cells (`), and B cells (z) were compared with that of a control MLR
without veto cells (䡺). The figure shows the veto effect of specific (origin
H-2d; A) and nonspecific (origin H-2q; B) freshly isolated cells. The results
represent the average ⫾ SD of three different experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a
Results expressed as a percentage of inhibition, calculated as follows: % inhibition ⫽ 1 ⫺ (frequency of 1B2⫹ CD8⫹ cells in the presence of veto cells)/(frequency
1B2⫹CD8⫹ cells in the absence of veto cells) ⫻ 100. For each cell subpopulation,
inhibition was tested in presence of specific cells of H-2d origin (A) or nonspecific
H-2s origin (B). Statistical significance was evaluated by Student’s t test.
In the present study we characterized several attributes of veto
cells as reflected by direct FACS measurement of H-2d-specific
effector T cells in the 2C TCR Tg mouse model. These attributes
include definition of the effective range of veto:effector ratios, the
role of cell contact and irradiation, and the role of cell activation.
Furthermore, by establishing the optimal conditions for this direct
assay it was possible to compare the veto reactivities of different
lymphocyte subpopulations on the same scale. Clearly, this direct
assay is not only more simple and straightforward, but it is also
less likely to be associated with artifacts typical of the indirect
lengthy functional assays. One major observation in the 2C model
is the lack of any appreciable veto activity exhibited by nonactivated lymphocytes. Thus, CD8⫹ or CD4⫹ T cells as well as NK
cells uniformly require prior activation to be endowed with veto
activity. In contrast, B cells do not exhibit veto activity even if
activated before testing. The importance of cell activation was previously indicated by an in vivo study in which CD8 T cells of male
origin were infused into female TCR Tg mice bearing a transgene
against the HY male Ag. Thus, although the infused veto cells
were able to induce reduction of the specific anti-male clones, this
activity was lost when using donor cells lacking the intracellular
domain of the CD8 molecule, important for the activation of CD8
T cells (16). Taken together, these indications pointing to the importance of cellular activation for the veto activity are intriguing
considering that freshly isolated (donor ⫻ host)F1 CD8⫹ T cells
were shown to facilitate bone marrow (35, 42, 43) or skin allografting (44). This discrepancy could be explained if these nonalloreactive F1 T cells were activated in vivo by a mechanism
similar to homeostasis, driven by the conditioning of the recipients
(45– 47). Alternatively, it is possible that freshly isolated CD8⫹ T
cells might induce tolerance to donor cells by a mechanism independent of the veto mechanism (48).
A second major finding of the present study is the establishment
of the relative hierarchy of veto activity, comparing anti-thirdparty CTLs, activated NK cells, and activated CD4⫹ T cells. The
former veto cell preparation was shown to be more effective by a
factor of 10 –20 compared with activated NK cells, which, in turn,
are more effective by a factor of 2.0 –50 compared with activated
CD4⫹ T cells. Interestingly, activated B cells are completely devoid of veto activity.
The role of NK cells in this context could be complex, because
it has been shown that alloreactive NK cells could potentially reduce host immune cells without GVHD, thereby enhancing engraftment of stem cell allografts (49 –51). Such ablation of hosttype immune cells is possibly also associated with undesirable
infections. However, the donor-specific veto reactivity measured
in the present study at different veto:effector ratios in the absence
6658
of alloreactivity (as evidenced by the control group in which NK
cells of a genetic background not recognized by the 2C effector
cells did not lead to deletion of the effector cells) strongly support
previous studies (23) suggesting that nonalloreactive NK cells
could induce tolerance with minimal ablation of host immunity.
It is well established that the level of immune debulking is inversely proportional to the number of veto cells required to overcome graft rejection. Thus, although megadose stem cell transplants could suffice to allow engraftment in heavily conditioned
patients, large numbers of other veto cells might be required to
establish stem cell engraftment in patients exposed to reduced intensity conditioning. Considering that new methodologies have
been developed to expand ex vivo NK cells (52–55) as well as host
nonreactive CD4⫹ T cells (56, 57), it might be possible in the
future to use all these different sources of veto cells to facilitate
engraftment of hemopoietic allografts without the unwanted complications of GVHD-producing T cells.
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