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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4038-4044
By
From the Division of Clinical Research, the Fred Hutchinson Cancer
Research Center; and the Department of Medicine, University of
Washington, Seattle, WA.
In allogeneic marrow transplantation, donor T cells that recognize
recipient alloantigens prevent rejection but also cause graft-versus-host disease (GVHD). To evaluate whether the ability to
prevent marrow graft rejection could be dissociated from the ability to
cause GVHD, we generated a panel of four different CD8 cytotoxic
T-lymphocyte clones specific for H2d alloantigens. Three of
the clones caused no overt toxicity when as many as 20 × 106 cells were infused intravenously into irradiated
H2d-positive recipients, and one clone caused acute lethal
toxicity within 1 to 3 days after transferring 10 × 106
cells into H2d-positive recipients. One clone that did not
cause toxicity was able to prevent rejection of (C57BL/6J ×
C3H/HeJ)F1 marrow in 800 cGy-irradiated (BALB/cJ × C57BL/6J)F1
recipients without causing GVHD. Large numbers of cells and exogenously
administered interleukin-2 were required to prevent rejection. These
results with different CD8 clones suggest that GVHD and prevention of
rejection could be separable effects mediated by distinct populations
of donor T cells that recognize recipient alloantigens.
THE RISK OF graft-versus-host disease
(GVHD) after allogeneic marrow transplantation can be decreased by
removing T cells from the donor marrow, but the use of T-cell depletion
has not improved disease-free survival because the benefit of decreased GVHD has been offset by an increased risk of graft failure and other
complications.1 GVHD is initiated by the relatively small subset of donor T cells that recognize recipient alloantigens. In
previous studies we have shown that donor T cells also prevent marrow
graft rejection most effectively when they recognize an alloantigen
expressed on recipient immune effectors that survive the pretransplant
conditioning regimen.2,3 Whether rejection is prevented
primarily by the same cells that cause GVHD is not known.
Recent studies have highlighted the role of proinflammatory cytokines
in the pathogenesis of GVHD.4 Donor T cells activated by
exposure to recipient alloantigens produce interferon- In the canine model, infusion of bulk-cultured cytotoxic T lymphocytes
(CTL) specific for recipient alloantigens helped to prevent major histocompatibility complex (MHC)-mismatched
marrow graft rejection, but all engrafted recipients died with acute GVHD.11 To determine whether prevention of rejection could
be separated from GVHD, we generated a panel of donor-derived CD8 CTL
clones specific for recipient alloantigens and tested their effects in
vivo in murine marrow transplant models. Among these clones, we
identified one that prevented allogeneic marrow graft rejection without
causing overt toxicity in the recipient. Differences among the clones
we tested suggest that the effector mechanisms involved in preventing
rejection might be distinct from those that cause GVHD.
Mice.
(C57BL/6J × SJL/J)F1 (B6SJL; H2b/s,
Ly5b/a) males, (C57BL/6J × C3H/HeJ)F1 (B6C3;
H2b/k) males, BALB/cJ (H2d,
Ly5b) females, B6.C-H2bm1/ByJ (bm1;
H2Kbm1, Ly5b) females, and
(BALB/cJ × C57BL/6J)F1 (CB6; H2d/b) females were
purchased from the Jackson Laboratory (Bar Harbor, ME).
B6.Ly5.1;pep3b (B6.Ly5a) males and
(C3H/HeJ × B6.Ly5a)F1
[(C3H × B6.Ly5a)F1; H2b/k,
Ly5a/b] males were bred at the Fred Hutchinson Cancer
Research Center (Seattle, WA). Founder B6.Ly5a males and
females were kindly provided by Dr David Myers (Sloan Kettering
Institute, New York, NY). Mice were housed in groups of five under
microisolated, specific pathogen-free conditions with twice weekly cage
changes and were administered sterilized chow and acidified water (pH
3.5) ad libitum. Four weeks after marrow transplantation, mice were
transferred to conventional housing conditions. Experimental procedures
were reviewed and approved by the Institutional Animal Care and Use
Committee of the Fred Hutchinson Cancer Research Center.
In vitro generation of CTL clones.
Responder B6C3 lymph node (LN) cells or splenocytes
(2.0 × 106/mL) were stimulated with irradiated (30 Gy)
CB6 or BALB/c spleen cells (1.0 × 106/mL) in culture
medium containing a 1:1 mixture of RPMI 1640 medium and enriched
Eagle's medium (Biofluids, Inc, Rockville, MD) supplemented with 10%
fetal calf serum (Hyclone, Logan, UT), 10 mmol/L L-glutamine, 100 U/mL
penicillin, 100 µg/mL streptomycin, 5 × 10 In vitro cytotoxicity assays.
Targets were prepared from LN cells activated with 2.0 µg/mL ConA and
then labeled with 51Cr. Cloned effectors and targets
(10 × 103/well) were incubated in RPMI 1640 medium
containing 5 mmol/L HEPES and 10% bovine serum for 4 hours at 37°C.
Results represent the means of triplicate determinations in which the
percent specific 51Cr release was calculated by standard
methods. Spontaneous release values were less than 30%.
Cytokine production.
Cloned T cells (1.0 × 106/well) were stimulated in
2.0-mL culture wells with immobilized CD3-specific antibody
145-2C1112 (hamster IgG; hybridoma kindly provided by Dr
Jeffrey Bluestone, University of Chicago, Chicago, IL). Culture
supernatants were collected and stored at Immunofluorescent staining.
Cells were stained with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE)-conjugated monoclonal antibodies specific for CD3
(145-2C11, hamster IgG),12 T-cell receptor (TCR)- Marrow transplantation.
Recipients 7 to 8 weeks of age were prepared by total body irradiation
in a single fraction from dual-opposed 60Co sources at an
exposure rate of 20 to 25 cGy/minute on the day before transplantation.
Marrow obtained by femur flush was depleted of T lymphocytes by rabbit
complement (1:10)-mediated lysis using a mixture of antibodies specific
for CD4, CD8, and Thy-1.2 (30-H12, rat IgG2b; hybridoma kindly provided
by Dr J.A. Ledbetter, Bristol-Myers-Squibb, Seattle, WA),19
each at optimal concentration. Nylon wool-nonadherent T lymphocytes
were obtained from pooled mesenteric, axillary, and femoral lymph
nodes. CD8 blasts were enriched from mixed lymphocyte cultures
(MLC) by complement-mediated lysis using a CD4-specific antibody. Mixtures containing T-cell-depleted donor marrow
(5.0 × 106 cells/recipient) and purified T cells, CD8
blasts, or cloned CTL were injected into recipients via the lateral
tail vein. In some experiments, recipients were administered
recombinant human IL-2 (Cetus Co, Emeryville, CA) intraperitoneally
(IP) on the day of transplant and for 6 to 13 consecutive days
thereafter. IL-2 doses are expressed as U of activity for supporting
the proliferation of murine CTLL-2 cells.
Prevention of rejection by CD8 blasts specific for recipient
alloantigens.
We have shown previously that donor CD8 cells isolated from lymph nodes
can prevent rejection of T-cell-depleted B6C3 marrow and cause GVHD in
irradiated (800 cGy) CB6 recipients.2 To determine whether
cultured T cells can have similar effects in vivo, CD8 cells were
recovered from 5-day mixed lymphocyte cultures in which B6C3 LN T cells
were stimulated with irradiated host-specific CB6 spleen cells and
tested for their ability to prevent allogeneic marrow graft rejection
and cause GVHD in CB6 recipients. Rejection of B6C3 marrow cells in CB6
recipients was prevented by CB6-specific CD8 blasts, and these cells
did not require exogenous IL-2 for optimal activity (Table
1). At least 2.5 × 105
CB6-specific CD8 blasts were needed to prevent rejection, suggesting that the cultured cells were substantially less effective than freshly
isolated LN cells which could prevent rejection with as few as 5.0 × 104 CD8 cells.2 Recipients transplanted with
grafts containing 1.0 to 1.25 × 106 freshly isolated LN
CD8 cells developed readily apparent GVHD manifested by weight loss
that began during the fourth week after transplantation as compared
with recipients transplanted with T-cell-depleted marrow alone (Fig
1). Recipients transplanted with grafts
containing 1.0 to 1.25 × 106 cultured CD8 blasts did not
develop overt GVHD manifested by weight loss when IL-2 was not
administered after the transplant (Fig 1). Cultured CD8 blasts did
cause weight loss in some experiments but not in others when IL-2 was
administered after the transplant (data not shown).
Testing of CD8 CTL clones.
To evaluate whether the ability to prevent marrow graft rejection could
be dissociated from the ability to cause GVHD, we generated a panel of
four different B6C3 CD8 CTL clones specific for H2d
alloantigens. Three of these clones caused no overt acute toxicity when
as many as 20 × 106 cells were infused
intravenously into irradiated CB6 recipients, even when exogenous IL-2
was administered to sustain viability and in vivo survival of the cells
(data not shown). One clone caused acute lethal toxicity within 1 to 3 days after transferring 10 × 106 cells into recipients
that expressed the alloantigen recognized by the clone. One clone
(designated 14C3) that did not cause acute toxicity was able to prevent
rejection of B6C3 marrow in CB6 recipients (Table
2). The 14C3 cells prevented rejection only
when IL-2 was administered after transplantation (Table 3, Exp
1), although there was some variation in
the minimum amount of IL-2 needed for this effect (Table 3, Exp 1 and
2). At least 20 × 106 14C3 cells were needed to prevent
rejection in this model (Table 3, Exp 3). Smaller numbers of 14C3 cells
could not prevent rejection even when large doses of IL-2 were
administered over an extended period of 13 days after transplantation
in an attempt to sustain survival of the clone in vivo (Table 3, Exp
3).
Expansion and decline of 14C3 cell numbers after transplantation.
To evaluate the persistence of 14C3 cells after transplantation,
irradiated CB6 recipients were transplanted with grafts containing 5.0 × 106 T-cell-depleted (B6.Ly5a × C3H)F1
marrow cells and 20 × 106 14C3 cells. Recipients were
treated with IL-2 (30,000 U IP) on the day of transplant and for 6 consecutive days thereafter. In two recipients tested on day 7 after
transplantation, 62% and 51% of LN cells were CD3-positive,
H2k-positive, and Ly5.1-negative, indicating that they
originated from the 14C3 clone and not from the Ly5.1-positive marrow
graft or from the H2k-negative recipient. On day 7, the
spleens in these two recipients respectively contained 3.4 × 106 and 2.6 × 106 14C3 cells. In two
recipients tested on day 14 after transplantation, LN suspensions
contained 10% and 4% 14C3 cells, and the spleens respectively
contained 0.3 × 106 and 0.2 × 106 14C3
cells. Thus, 14C3 cells were able to traffic through lymphoid organs
and persist for at least 2 weeks after transplantation, but the number
of 14C3 cells decreased after treatment with IL-2 was discontinued on
day 7 after transplantation. Longer periods of IL-2 administration did
not prevent the decline in numbers of 14C3 cells in lymph nodes and the
spleen after day 7 (data not shown), suggesting that in vivo survival
of 14C3 cells was limited by factors other than the availability of
IL-2.
Functional characteristics of CTL clones.
The 14C3 clone and the other two clones that did not cause acute lethal
toxicity had similar characteristics, despite the differences in
ability to prevent marrow graft rejection. All three clones expressed
CD3 and TCR-
Prevention of rejection by 14C3 cells requires recognition of a
recipient alloantigen.
Three donor/recipient strain combinations were tested to determine the
recognition requirements for prevention of marrow graft rejection by
14C3 cells. With B6C3 donors and CB6 recipients, 14C3 cells recognize
an alloantigen expressed by recipient T cells (H2Kd).
Rejection of B6C3 marrow in 800 cGy irradiated CB6 recipients was
prevented by adding 2.5 × 105 B6C3 LN T cells to the
graft and also by adding 20 × 106 B6C3-derived 14C3 cells
to the graft (Table 5), confirming results shown in Tables 2 and 3. With B6SJL donors and CB6 recipients, 14C3
cells recognize an alloantigen expressed by recipient T cells (H2Kd), but they do not express H2s
alloantigens that provoke rejection of the B6SJL marrow graft and
therefore cannot prevent rejection by interfering with the generation
of cytotoxic responses against H2s alloantigens or by other
passive mechanisms. Rejection of B6SJL marrow in 800 cGy irradiated CB6
recipients was prevented by adding 1.0 × 106
B6SJL LN T cells to the graft and also by adding 20 × 106
B6C3-derived 14C3 cells to the graft (Table 5). With
B6.Ly5a donors and bm1 recipients, 14C3 cells do not
recognize any alloantigens expressed by recipient T cells, but they do
express the H2Kb alloantigens that provoke
T-cell-mediated22 rejection of the B6.Ly5a
marrow graft. Rejection of B6.Ly5a marrow in 550 cGy
irradiated bm1 recipients was prevented by adding 2.5 × 105 B6.Ly5a LN T cells to the graft but not by
adding 20 × 106 14C3 cells to the graft (Table 5).
Results with B6.Ly5a donors and bm1 recipients were similar
when 14C3 cells were activated either by stimulation with immobilized
CD3-specific antibody for 1 day before transplantation or by adding 5.0 × 106 unirradiated T-cell-depleted CB6 marrow cells to
the graft. Taken together, these results show that 14C3 must recognize
an alloantigen on recipient cells to prevent marrow graft rejection.
Evaluation of toxicity caused by infusion of 14C3 cells in allogeneic
recipients.
The ability of 14C3 cells to cause GVHD through recognition of
H2Kd in irradiated CB6 recipients was evaluated by
monitoring weight and by histological examination after transplantation
of T-cell-depleted B6C3 marrow. During the first week after
transplantation, recipients treated with 14C3 cells had slightly more
weight loss than negative controls transplanted with grafts containing
no added T cells (P < .001) (Fig
2). After the first week, 14C3 recipients
recovered completely and showed a weight gain profile identical to that of negative controls, whereas positive controls transplanted with grafts containing 2.5 × 105 LN T cells developed GVHD
manifested by weight loss that began during the third week after
transplantation as compared with recipients transplanted with
T-cell-depleted marrow alone (P < .005 at day 21, and
P < .001 at day 28). The transient weight loss caused by
14C3 cells was not accompanied by histological changes diagnostic of
GVHD in the skin, liver, gut, or lung of recipients evaluated on day 7 after transplantation (data not shown). Increased weight loss during
the first week after transplantation also occurred when 14C3 cells were
administered with T-cell-depleted B6SJL marrow in CB6 recipients but
not when 14C3 cells were administered with B6.Ly5a marrow
in bm1 recipients (data not shown). Thus, weight loss did not occur in
recipients lacking the H2Kd alloantigen recognized by 14C3
cells.
Results in the present study have shown two notable findings. First,
bulk-cultured, alloantigen-stimulated CD8 cells and certain cloned CD8
cytotoxic effector cells caused little or no overt acute toxicity after
adoptive transfer into irradiated recipients expressing antigen(s)
recognized by the CTL. Second, one of the clones we generated was able
to prevent allogeneic marrow graft rejection without causing GVHD.
These results suggest that GVHD and prevention of rejection could be
separable functional effects mediated by distinct populations of donor
T cells that recognize recipient alloantigens.
Submitted October 20, 1997;
accepted January 20, 1998.
The experiments described in this study were performed with assistance
from Kelli McIntyre, and the manuscript was prepared with assistance
from Alison Sell. The authors thank Dr Michael A. Bean for helpful
discussions and Dr Martin A. Cheever and Dr Ilonna Rimm for critical
reading of the manuscript.
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Abstract
Introduction
Abstract
(IFN-
(TNF-
5 mol/L
2-mercaptoethanol, and 5 mmol/L HEPES. Cultures were fed with fresh
medium on the 5th day, and cells (1.0 × 106/well) were
restimulated on the 10th day with irradiated (30 Gy) allogeneic
splenocytes (5 × 105/well) in the presence of
irradiated (30 Gy) syngeneic (responder strain) splenocytes
(5 × 106/well) as filler cells in 24-well plates (2.0 mL/well). Three days after each stimulation, cultures were split 1:4
with fresh medium containing 5 U/mL human recombinant interleukin-2
(IL-2; Cetus, Emeryville, CA). After repeated cycles of restimulation at 10 to 21 day intervals, cells were cloned by limiting dilution in
medium containing 10 U/mL IL-2 and 10 ng/mL murine recombinant IL-7
(R & D Systems, Minneapolis, MN), with irradiated (30 Gy) syngeneic
splenocytes used as filler cells. T-cell clones were propagated by
stimulation with irradiated allogeneic splenocytes (5 × 105/well) in the presence of irradiated (30 Gy)
syngeneic splenocytes (5 × 106/well), 10 U/mL IL-2, and
1 to 10 ng/mL IL-7. T-cell clones were rested for at least 14 days
between cycles of restimulation and for at least 7 days before testing
in vitro or in vivo unless otherwise indicated.
20°C until testing.
IL-2/IL-4 activity was determined by a biological assay using
CTLL-2 cells, and other cytokines were measured with
enzyme-linked immunosorbent assay kits and standards from Genzyme
(Cambridge, MA) (IFN-
(H57-597, hamster IgG; Pharmingen, San Diego, CA),
(GL3, hamster IgG; Pharmingen),
C3H/HeJ) and (C3H/HeJ