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*
Department of Pediatrics, Division of Bone Marrow Transplantation, and
Department of Therapeutic Radiology, University of Minnesota Cancer Center, Minneapolis, MN 55455; and
Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Graft-versus-host disease (GVHD) is caused by donor T cells that are alloactivated in vivo upon contact with host alloantigen-bearing target cells. Previously, we have shown that the administration of either CTLA4-Ig fusion protein or anti-B7.1 + anti-B7.2 (anti-B7) mAb can down-regulate GVHD-induced mortality and inhibit donor T cell expansion in vivo (17, 18, 19, 20). We also have observed that the in vivo administration of anti-B7 mAb completely eliminates the graft-versus-leukemia (GVL) effect of delayed donor lymphocyte infusion (DLI) given to allogeneic BMT recipients that were subsequently challenged with acute myeloid leukemia tumor cells 1 wk after splenocyte infusion (21). Because CD28:B7 and CTLA-4:B7 interactions may counterbalance each other, we sought to determine the separate and potentially interrelated roles of each pathway in regulating donor T cell expansion, activation, and function in vitro and in vivo. In this study, we demonstrate that CD28:B7 and CTLA-4:B7 interactions have contrasting effects on the expansion of donor anti-host (GVHD-causing) and host anti-donor (graft-rejecting) T cell responses. These data suggest that the optimal exploitation of competitive antagonists to these pathways will depend upon the circumstances and timing of administration post-BMT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.BR/SgSnJ (H2k), C57BL/6 (termed B6:H2b,CD45.2), CD28-deficient B6 (CD28-/-), C.H2bm1 (termed bm1; CD45.2), and C.H2bm12 (termed bm12; CD45.2) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). BALB/c-SCID (H2d) and B6-CD45.1 congenic mice were purchased from the National Institutes of Health (Bethesda, MD). Mice were housed in a specific pathogen-free facility in microisolator cages. Donors and recipients were used at 8–10 wk of age.
GVHD generation
To determine the effect of CD28/CTLA-4:B7 interactions on the
GVHD capacity of CD4+ or CD8+ T cells, we used
a system in which purified T cell subsets are given to MHC disparate
sublethally irradiated recipients, as initially described by Sprent
(22). This system permits highly accurate quantification of the degree
of GVHD responses as related to T cell dose. For this purpose, MHC
class II (bm12) or class I (bm1) disparate recipients were irradiated
with 6 Gray (Gy) TBI on day 0 from a 137Cesium source at a
dose rate of 85 cGy/min (20, 22). Four to six hours after TBI,
sublethally irradiated bm12 recipients were given purified lymph node
(LN) B6CD4+ T cells at doses of 0.3–3 x
105 per recipient, while bm1 recipients were injected with
CD8+ T cells at doses of 0.3, 1, or 3 x
106 per recipient. To purify LN cells, single cell
suspensions of axillary, mesenteric, and inguinal LN cells were
obtained (as a source of GVHD-causing effector cells), depleted of NK
cells, and enriched for CD4+ or CD8+ T cells by
depletions with anti-CD8 (hybridoma 2.43, rat IgG2b, provided by
Dr. David Sachs, Charlestown, MA) or anti-CD4 (hybridoma GK1.5, rat
IgG2b, provided by Dr. Frank Fitch, Chicago, IL), respectively. Rat
mAb-coated T cells were passaged through a goat anti-mouse and goat
anti-rat Ig-coated column (Biotex, Edmonton, Canada). The final
composition of T cells in the donor graft was determined by flow
cytometry and was always found to be 
94% T cells of the desired
phenotype. Hematocrit values were obtained at periodic intervals as an
indicator of the possible BM destructive effects of infused T cells
(20, 22). For all GVHD and engraftment systems, mice were monitored
daily for survival and clinical appearance and weighed twice weekly.
To avoid a host anti-donor response present in the sublethal irradiation systems, we employed two different types of strategies. In the first, mice were lethally irradiated with 8 Gy TBI by x-ray (39 cGy/min) on day -1, followed on day 0 by the i.v. infusion of T cell-depleted (TCD) BM by anti-Thy-1.2 (clone 30-H-12, provided by Dr. David Sachs, Cambridge, MA) + complement treatment. To measure CD4+ and CD8+ T cell GVHD responses, irradiated B10.BR recipients were infused i.v. with 20 x 106 TCD BM along with 0 or 15 x 106 splenocytes from B6 or CD28-/- donors. To measure CD4+ T cell GVHD responses, irradiated bm12 recipients were infused with 8 x 106 TCD BM along with 0 or 0.3 x 106 purified T cells from B6 or CD28-/- donors. In a second system designed to analyze the effect of CD28-/- T cells in the complete absence of tissue damage induced by high dose TBI, BALB/c-SCID recipients were treated with anti-asialo GM1 antisera (25 µl on days -4 and -2) to deplete endogenous NK activity and then infused with 2 x 106 LN T cells from B6 or CD28-/- donors without further conditioning of the recipient.
Thoracic duct cannulation
For thoracic duct lymphocyte (TDL) isolation, cannulae were inserted in the thoracic duct of recipients at the time of peak proliferation (day 6) post-BMT. TDL were collected over a period of 18 h (20).
GVL studies
Our GVL induction procedure has been described in detail (21). In brief, mice undergoing BMT for GVL assessment were conditioned with 800 cGy irradiation from an x-ray source on day -1 and infused with 8 x 106 B10.BR TCD BM on day 0. Donor B10.BR splenocytes were administered i.v. at a dose of 25 x 106 on day 21 post-BMT. On day 28 post-BMT, mice were challenged with an MHC class I+II- acute myeloid leukemia line, C1498 (21). C1498 (American Type Culture Collection, Manassas, VA), originally derived as a spontaneous tumor line from a B6 mouse, was grown in RPMI (Life Technologies, Grand Island, NY) with 10% heat-inactivated FBS (HyClone, Ogden, UT), 2 mmol/L L-glutamine, 50 mmol/L 2-ME, and penicillin/streptomycin. C1498 cells (2 x 105) were given via the i.v. route. All available animals were examined for evidence of GVHD and tumor by necropsy (21).
Engraftment studies
B6-CD45.1 mice were irradiated with 6 Gy TBI by x-ray on day -1 and then given bm1 or bm12 TCD BM (10 x 106) cells (23). Peripheral blood cells were phenotyped for chimerism twice post-BMT. In addition, the presence of donor or host origin cells in individual lineages was analyzed in detail at least once post-BMT.
Anti-CTLA-4 mAb preparation
Clone UC10-4F10-11 (hamster anti-murine CTLA-4) was generously provided by Dr. Jeffrey Bluestone (University of Chicago, Chicago, IL) (9). mAb for injection was generated as ascites fluid and purified by ammonium sulfate precipitation, followed by dialysis. Control hamster IgG was purchased from Rockland Laboratories (Gilbertsville, PA). Cohorts of mice were given anti-CTLA-4 mAb or irrelevant hamster IgG mAb. In initial GVHD studies, anti-CTLA-4 mAb or control mAb was given at a dose of 250 µg/dose on days -1 and 0, and then 100 µg/dose three times per week until day 10. In a subsequent study, a more intensive regimen was used consisting of anti-CTLA-4 mAb or control mAb at 400 µg/dose from days -1 to +5, then 200 µg/dose three times a week until day 14 (for engraftment studies) or day 21 (for GVHD studies) or day 24 after delayed lymphocyte infusion (for GVL studies).
Flow cytometry
The T cell, B cell, and granulocyte/macrophage constituency of
splenocytes, peripheral blood cells, or TDL effluent were measured
using mAb directed toward CD4 or CD8, CD19, and Mac1, respectively. All
fluorochrome-labeled mAb, unless indicated, were obtained from
PharMingen (San Diego, CA). For bm1 recpients of B6-CD45.1
CD8+ T cells, splenocyte suspensions were monitored with
CD45.2 (clone 104-2, rat IgG2a) and CD45.1 (clone A20-1.7, rat
IgG2a), both provided by Dr. U. Hammerling (New York, NY). For splenic
flow cytometry studies, cells were first incubated with 2.4G2 to block
Fc receptors, and then incubated with an optimal concentration of
fluorochrome-labeled mAb for 45 min at 4°C. Cells were washed three
times and resuspended for analysis by two- or three-color flow
cytometry using FITC-, PE-, or biotin (along with
streptavidin-PerCP)-conjugated mAb purchased from PharMingen or
Becton Dickinson (Mountain View, CA). Irrelevant mAb control values
were subtracted from values obtained with relevant mAbs. All results
were obtained using a FACScalibur (Becton Dickinson). Forward and side
scatter settings were gated to exclude red cells and debris, and 1
x 104 cells were analyzed for each determination.
In vitro MLR cultures and CTL assessment
For quantifying MLR responses, CD4+ LN T cells or
TDL were mixed with irradiated (30 Gy) TCD splenocyte stimulators from
host strain mice. Cells were suspended in MLR media consisting of
Dulbecco’s MEM (Bio Whittaker, Walkersville, MD), 10% FCS (HyClone,
Logan, UT), 2-ME (5 x 10-5 M) (Sigma, St. Louis,
MO), 10 mM HEPES buffer, 1 mM sodium pyruvate (Life Technologies, Grand
Island, NY), amino acid supplements (1.5 mM L-glutamine,
L-arginine, and L-asparagine) (Sigma), and
antibiotics (penicillin, 100 U/ml; streptomycin, 100 µg/ml) (Sigma).
A total of 0.3–1 x 105 cell responders and 1 x
105 irradiated stimulators were plated into 96-well
round-bottom (Costar) plates and placed at 37°C and 10%
C02 for 1 to 6 days. In some wells, as indicated,
anti-CTLA-4 mAb was added at a final concentration of 50 µg/ml.
cpm were calculated by subtracting the syngeneic proliferative
response from the allogeneic proliferative response. No scintillant was
used to amplify cpm detection. For CTL analysis, splenocytes were
activated with Con A (2 µg/ml) for 3 days in MLR media and then
labeled overnight with [3H]thymidine (4 µCi/ml) in
24-well plates containing 2 x 106 cells in a 2 ml
vol. B6 or CD28-/- TDL T cells were spun at 500 rpm
(Beckman TJ-6) for 5 min with targets (104 targets/well) at
E:T ratios of 12.5:1–100:1 in MLR media and then incubated together
for a period of 4 h. Cell pellets were harvested and then counted
as described above for the MLR culture. The percent specific lysis was
based upon the retention of [3H]thymidine in the
experimental groups as compared with targets not exposed to effector
cells (24).
In situ antisense:sense mRNA hybridization of TDL
TDL cytospins were hybridized with riboprobes specific for IL-2
237–837(237–837), IL-4 1–393(1–393), IL-5 1–1534(1–1534), IFN-
1–270(1–270), IL-10
392–937(392–937), granzyme B 239–775(239–775), and perforin 1–1700(1–1700) mRNA. The
mRNA:RNA probe hybrids were detected using an alkaline phosphatase
-digoxigenin F(ab')2 Ab and BCIP/NBT
(5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) color
substrate. The in situ hybridization technique has been described
elsewhere (25).
Statistical analyses
Group comparisons of continuous data were made by Student’s
t test. Survival data were analyzed by lifetable methods
using the Mantel-Peto-Cox summary of 
2 (26). Actuarial
survival and relapse rates are shown. p values < 0.05
were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blockade of CD28/CTLA-4:B7 interactions by CTLA4-Ig or anti-B7 mAb has been shown to diminish GVH alloresponses, resulting in a decrease in GVHD-induced mortality in some studies (17, 18, 19, 20, 27, 28, 29, 30). Because CTLA4-Ig and anti-B7 mAb inhibit both the CD28:B7 and CTLA-4:B7 interactions, the analysis of the contributions of each pathway individually in GVHD generation has required alternative approaches, including the use of CD28-/- mice. To determine whether precluding CD28:B7 interactions would have a beneficial effect on preventing GVH lethality, highly purified wild-type or CD28-/- T cells were infused into sublethally irradiated wild-type or MHC disparate recipients. In this setting, alloreactive donor T cells cause multiorgan system GVH tissue destruction (20), and because the hemopoietic compartment is a target of alloresponsive donor T cells, recipients frequently experience lethal BM aplasia. A major advantage of this type of system includes the close correlation between T cell dose and lethality rates.
To quantify the effect of CD28:B7 interactions on GVH lethality of
CD4+ T cells, CD28-/- CD4+ T
cells were infused at doses of 0.3–3 x 105 into
sublethally irradiated bm12 (MHC class II only disparate) recipients.
At all cell doses tested, CD28-/- CD4+ T
cells were found to be incapable of causing GVHD lethality (Fig. 1
A). In contrast, recipients
of as few as 0.3 x 105 wild-type cells had a 70%
lethality rate, while recipients of 1 or 3 x 105
wild-type cells uniformly succumbed to GVHD. Weight loss was observed
in recipients of wild-type, but not CD28-/- cells,
reflective of survival results. Day 14 mean hematocrit values were
17% in recipients of 105 wild-type T cells that were
significantly lower than the mean values of 25% in recipients of
0.3 x 105 wild-type T cells or 32% obtained from
recipients of CD28-/- T cells.
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B).
As few as 0.3 x 106 wild-type cells resulted in 69%
lethality and 106 cells were 100% lethal. Weight curves
were reflective of survival. Day 14 hematocrit values were
significantly lower (17%) in recipients of wild-type cells as
compared with recipients of CD28-/- T cells (27%).
Based upon these data, there was at least a 10-fold reduction in the
GVH lethality capacity of CD28-/- CD4+ T
cells and >3- but <10-fold reduction in the GVH lethality of
CD28-/- CD8+ T cells.
To determine whether CD4+ and CD8+ T
cell-containing inocula from CD28-/- donors were capable
of causing GVHD, we used a different model system in which the B6
(H2b) donor and B10.BR (H2k) recipient were
mismatched at both MHC class I and class II loci. For this purpose and
to simulate a clinical BMT setting in which there is no host
anti-donor response as would be present in sublethally irradiated
recipients, we used a model system in which the recipient is lethally
irradiated and then given 5 or 15 x 106 B6
splenocytes along with TCD BM (Fig. 1C). Recipients of the
higher numbers of CD28-/- had a significantly higher
actuarial survival rate as compared with wild-type controls, although
both groups ultimately succumbed to GVHD lethality. The degree of GVHD
protection was <3-fold since recipients of 15 x 106
CD28-/- splenocytes had a significantly lower survival
rate than recipients of 5 x 106 wild-type cells.
Recipients of as few as 5 x 106 CD28-/-
cells had a high survival rate (80% at 100 days), but also had
clinical and weight evidence of sublethal GVHD.
The lower degree of GVHD protection could have been due to the use of
more irradiation in the B6
B10.BR model than in the sublethal system.
To investigate this possibility, we chose a different system in which
donor CD4+ and CD8+ T cells are infused into an
MHC class I and class II disparate recipient (BALB/c-SCID) that does
not require any TBI conditioning of the recipient to permit donor T
cell-mediated GVHD lethality. NK-depleted BALB/c-SCID mice were given
2 x 106 T cells from B6 or CD28-/-
donors. Ninety percent of recipients of either CD28-/- or
wild-type cells succumbed to GVHD mortality within 1 mo after transfer,
with both groups averaging >30% loss in mean body weights (data not
shown). Together, these data suggest that the reduced GVHD response
observed when low numbers of CD28-/- CD4+ or
CD8+ T cells are infused into sublethally irradiated
recipients can be overwhelmed when larger numbers of donor
CD28-/- CD4+ and CD8+ T cells are
coinfused into MHC disparate recipients.
Blockade of CTLA-4:B7 interactions increases donor T cell expansion and GVHD lethality, as well as host-mediated BM graft rejection early post-BMT
The reduced GVHD lethality observed with CD28-/- T cells in sublethally irradiated recipients could be due to reduced expansion of CD28-/- T cells due to the absence of a positive regulatory signal or to unperturbed CTLA-4:B7 interactions due to the presence of a negative regulatory signal. In the latter instance, CTLA-4:B7 interactions might be expected to down-regulate donor T cell responses. To directly investigate the latter possibility, anti-CTLA-4 mAb was given in vivo to block CTLA-4:B7 interactions. To address the possibility that anti-CTLA-4 mAb could directly affect donor anti-host T cell alloresponses, in vivo donor T cell expansion and function and GVHD mortality were quantified.
To quantify donor T cell expansion unencumbered by a host
anti-donor T cell response, BALB/c-SCID recipients were depleted of
NK cells and then given 0.2 x 106 or 2 x
106 T cells from (B6 x 129)F2
(H2b) donors. On days 4 and 6 posttransfer, the number of
TDL was quantified in recipients of 2 x 106 cells as
an indicator of donor T cell expansion in vivo. We chose to isolate TDL
rather than study splenic T cells since more donor T cells are
collectable in the lymphatics over an 18-h period than present in the
spleen and because splenic but not TDL T cells reside in an environment
that inhibits the generation of an immune response. At both time
periods, recipients of anti-CTLA-4 mAb had a 2-fold increase in TDL
T cell number as compared with those that received irrelevant mAb (day
4, 0.295 x 106/ml vs 0.155 x
106/ml; day 6, 8.14 x 106/ml vs 4.17
x 106/ml, respectively) (data not shown). In a different
experiment, recipients were given lower numbers of T cells (0.2 x
106) and cannulated on day 9 posttransfer. Once again, the
number of TDL was 2-fold higher in anti-CTLA-4 mAb-treated as
compared with irrelevant mAb-treated controls (1.09 x
106/ml vs 0.49 x 106/ml, respectively)
(data not shown). Phenotypic analysis of days 4, 6, and 9 TDL cells
showed no substantial differences in either the proportion of
CD4+ and CD8+ T cells or the expression of a
broad panel of activation Ags (data not shown). Proliferative responses
to host stimulator cells were measured using day 6 posttransfer TDL
from irrelevant or anti-CTLA-4 mAb-treated recipients. In both
experiments, peak responses to host stimulator cells were seen after 1
day of culture using TDL obtained from both cohorts as compared with
the typical 5 days required for peak response using TDL obtained from
non-BMT donor-strain controls. Results from one of these two replicate
experiments (Fig. 2) are consistent with
an in vivo priming effect. In situ hybridization analysis for cytokine
mRNA in fresh day 6 TDL obtained from a pool of four to five mice in
each group indicated that anti-CTLA-4 mAb led to a skewed Th2
response, as indicated by an increase in the percentages of TDL cells
expressing IL-4 mRNA (1.2% vs 16.4%, respectively), although the
percentage of IL-10 mRNA-expressing cells remained similar. The
percentage of TDL cells expressing Th1 cytokines (IL-2, IFN-) was
low in both the control and anti-CTLA-4 mAb groups (<0.3%). The
frequency of TDL cells expressing granzyme B was comparable (12.3%
vs 11.4%, respectively).
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). Weight curves (data not shown) and
clinical appearance of recipients were reflective of survival curves,
consistent with a fulminant GVHD process in anti-CTLA-4 mAb-treated
recipients. These findings were reproduced in a second experiment.
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Donor TCD BM was infused in sublethally irradiated recipients of MHC
class I (bm1B6) or II (bm12B6) disparate recipients. Peripheral
blood chimerism of T, B, and myeloid lineages was assessed at time
periods up to 145 days post-BMT. In both strain combinations,
recipients given anti-CTLA-4 mAb had significantly lower
proportions of donor cells and higher proportions of host cells, with
all lineages being affected (Table I).
Particularly striking were the increases in the proportion of host
CD4+ and CD8+ T cells in the anti-CTLA-4
mAb-treated recipients, indicating that anti-CTLA-4 mAb stimulates
a host anti-donor response culminating in a higher degree of graft
resistance. These data provide a likely explanation for the improved
survival rate in sublethally irradiated recipients of allogeneic T
cells and anti-CTLA-4 mAb (data not shown), which is presumably due
to an augmented host anti-donor resistance process, limiting the
GVH lethality effect of donor T cells. Together with the GVH results,
these data indicate that the selective targeting of CD28:B7
rather than targeting both CD28:B7 and CTLA-4:B7 interactions would be
desirable in preventing GVH lethality and preserving alloengraftment
early post-BMT.
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The enhanced donor T cell expansion caused by anti-CTLA-4 mAb could be advantageous in augmenting the GVL effect of donor T cells given post-BMT. Because anti-CTLA-4 mAb increases GVHD lethality when given early post-BMT, we elected to study the potential GVL effect of anti-CTLA-4 mAb given later post-BMT when GVHD lethality of donor T cells is markedly reduced. For this purpose, we utilized a model of delayed splenocyte infusion in which lethally irradiated B6 recipients are reconstituted with B10.BR TCD BM for a period of 3 wk before donor splenocyte infusion (21). Donor T cells given at this later time period post-BMT have a markedly lower capacity to cause GVHD lethality than when given on day 0 of BMT (21). One week after donor splenocyte infusion, recipients are challenged with a supralethal dose of AML cells of the B6 strain. In previous studies, we have shown that the GVL effects of delayed splenocyte infusion against AML cells in this model are dependent upon the binding of B7 ligand(s) to T cell counter-receptor(s) (21). Donor CD8+ T cells and, to a lesser extent, CD4+ T cells present in the donor spleen cells are responsible for the GVL effect of DLI in this setting, while NK cells do not substantially contribute to this response (21).
To discern whether blockade of CTLA-4:B7 binding would enhance the GVL
effect against AML cells, anti-CTLA-4 mAb was given to
reconstituted recipients of delayed post-BMT donor splenocyte infusion,
which are challenged with AML cells. Donor splenocytes provided a GVL
effect that delayed the occurrence of leukemia as compared with
recipients not given donor splenocytes (Fig. 4A), although all mice treated
with donor splenocytes eventually succumbed with leukemia by day 75
post-BMT. In striking contrast, none of the anti-CTLA-4 mAb-treated
recipients of donor splenocytes and AML cells examined during the
entire 220-day risk period had evidence of leukemia, despite the fact
that donor splenocyte infusion alone did not prevent leukemia
occurrence in any of the recipients analyzed 75 days post-BMT.
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B). Anti-CTLA-4 mAb-treated recipients had a tendency
toward lower weights than controls that received neither AML cells nor
anti-CTLA-4 mAb (data not shown). Of note, the 100-day GVHD
mortality risk, presumably the greatest risk period for observing
mAb-induced GVHD acceleration of donor splenocytes, was highly
comparable with survival rates of 44% or 50% in these two groups,
respectively. Taken together, these data indicate that anti-CTLA-4
mAb given later post-BMT in the context of DLI mediated a potent GVL
effect, while at most only slightly increasing the GVH side effects of
DLI. Role of CTLA-4:B7 interactions on modulating CD28-dependent and CD28-independent T cell responses
CTLA-4:B7 interaction may serve to regulate CD28:B7 responses.
Therefore, experiments were performed to examine the role of CTLA-4:B7
in modifying the responses of CD28-/- T cells to
alloantigens. We first examined CD4+ T cell responses
obtained from wild-type or CD28-/- donors on in vitro
proliferative responses (Fig. 5A) and in vivo GVH lethality
responses (Fig. 5B) to MHC class II disparate bm12 strain
mice in the presence or absence of anti-CTLA-4 mAb. In vitro
proliferative responses of CD28-/- CD4+ T
cells to bm12 stimulator cells were lower than observed using
CD4+ T cells from wild-type controls beginning on day 2 of
culture (Fig. 5A). Anti-CTLA-4 mAb increased proliferation
of wild-type but not CD28-/- T cells.
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B). In contrast, recipients given
CD4+ T cells from CD28-/- donors had 100%
survival regardless as to whether these mice received irrelevant or
anti-CTLA-4 mAb. To attempt to uncover a more modest effect of
anti-CTLA-4 mAb on accelerating GVHD lethality of
CD28-/- CD4+ T cells, the number of donor T
cells was increased to 106 per recipient. Under these
conditions, acceleration of GVHD by anti-CTLA-4 mAb given to
recipients of wild-type CD4+ T cells was difficult to
assess since these recipients all succumbed by day 10 post-BMT.
Anti-CTLA-4 mAb-treated recipients uniformly died by day 9 post-BMT.
Recipients (n = 10/group) given 106
CD4+ T cells from CD28-/- donors and
irrelevant mAb had an 80% long-term (>2.5-mo post-BMT) survival rate
(Fig. 5C). Anti-CTLA-4 mAb-treated recipients had a 50%
survival rate that was of borderline (p = 0.08)
significance. Because recipients of a 3-fold higher number of
CD28-/- CD4+ T cells (3 x
106) had 0% survival, these data would indicate that
anti-CTLA-4 mAb infusion resulted in <3-fold increase in
CD28-/- CD4+ T cell-mediated lethality.
To determine whether alloreactive CD28-/-
CD4+ and CD8+ T cells would be stimulated to
expand in the presence of anti-CTLA-4 mAb, TDL were obtained from
cohorts of lethally irradiated B10.BR recipients of B6 or
CD28-/- donor splenocytes on days 6 and 9 post-BMT. At
the time of peak expansion (day 6), donor T cell expansion was
significantly higher in recipients of wild-type as compared with
CD28-/- donor cells (Fig. 6A). The increase in TDL T
cell number in anti-CTLA-4 mAb-treated BMT recipients was CD28
dependent since no increases in TDL T cells were observed in
mAb-treated recipients of CD28-/- T cells. Thus, under
these conditions, the negative regulatory role of CTLA-4:B7 interaction
is CD28 dependent. Flow cytometry analysis of TDL obtained on days 6
and 9 did not reveal any obvious differences in activation profiles
(data not shown). In recipients of wild-type and CD28-/-
donor splenocytes, donor CD4+ T cells comprised a higher
proportion of the TDL on days 6 and 9 post-BMT in recipients given
anti-CTLA-4 mAb as compared with irrelevant hamster IgG mAb (50%
increase over baseline percentages at both time points). In situ
hybridization analysis for cytokine mRNA was performed using fresh TDL
obtained from a pool of four mice per group. In recipients of
CD28-/- T cells, TDL were skewed toward a Th2 phenotype,
as evidenced especially by increases in the frequency of IL-5
(5.7-fold) and IL-10 (5.5-fold), with a concomitant decrease in IFN-
mRNA-expressing cells (Table II). In
addition, as observed in the (B6 x
129)F2BALB/c-SCID system, anti-CTLA-4 mAb
administration led to a skewed Th2 response in TDL obtained from
recipients of wild-type cells, and further skewed the Th2 response of
CD28-/- T cells increasing the frequency of TDL cells
expressing IL-4, IL-5, and IL-10 (2.5-fold), while decreasing the
frequency of IFN--expressing cells to frequencies that were 3-fold
to 6-fold lower than controls. Also as previously noted,
anti-CTLA-4 mAb did not substantially alter the percentage of
granzyme B and perforin mRNA-expressing TDL T cells (Table II).
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B). Peak
responses in all groups on day 1 were indicative of in vivo priming.
Wild-type TDL had greater proliferation to stimulator cells than
CD28-/- TDL. Proliferative responses of TDL obtained from
anti-CTLA-4 mAb-treated recipients were not higher than observed
with TDL from irrelevant mAb-treated recipients. Cytolysis against host
target cells was higher when using CD28-/- T cells as
effector cells (Fig. 6C). Anti-CTLA-4 mAb did not increase
the cytolytic capacity of TDL obtained from either irrelevant or
anti-CTLA-4 mAb-treated recipients, despite the fact that the
proportion of CD8+ T cells was lower in TDL obtained from
anti-CTLA-4 mAb versus irrelevant mAb-treated recipients.
Therefore, although CTLA-4:B7 regulates the expansion of wild-type but
not CD28-/- T cells, neither the in vitro anti-host
proliferative nor cytolytic responses of TDL T cells from recipients of
wild-type or CD28-/- appear to be substantially increased
by anti-CTLA-4 mAb infusion. |
Discussion |
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Blockade of CD28/CTLA-4:B7 interactions by CTLA4-Ig or anti-B7 mAb has been shown to diminish GVH alloresponses, resulting in a decrease in GVHD-induced mortality in some studies (14, 15, 16, 17, 27, 28, 29, 30, 31, 32). Because CTLA4-Ig and anti-B7 mAb preclude both the CD28:B7 and CTLA-4:B7 interaction, the separate contributions of these pathways in GVHD generation have required alternative approaches, including the use of CD28-/- mice. In vitro, alloresponses of CD28-/- T cells are markedly lower than controls. Indeed, CD28-/- CD4+ or CD8+ T cells have a reduced GVHD lethality capacity or >10-fold and >3-fold, respectively, when infused into sublethally irradiated recipients, confirming and extending studies of Anasetti and coworkers (32). In some experiments in the strain combinations used for our studies, differences in donor T cell expansion were sufficient to provide a significant degree of protection from GVHD lethality, especially under situations in which the onset of lethal GVHD was not rapid. However, in other strain combinations in which recipients uniformly succumbed within 10–22 days following allogeneic donor T cell infusion, CD28:B7 interaction was not necessary for optimal GVH lethality. Others have shown either a slight or no delay in GVHD-induced lethality in sublethally irradiated recipients given high numbers of allogeneic CD28-/- donor splenocytes (30) or a markedly diminished mortality rate, but with significant histologic GVHD in heavily irradiated F1 recipients of CD28-/- parental T cells (31). As measured by thoracic duct cannulation, allogeneic CD28-/- T cells will expand in vivo, albeit to a far lesser extent than wild-type cells. Similar conclusions were reached by Anasetti and coworkers, who analyzed the spleens of unirradiated F1 recipients of CD28-/- donor spleen cells (32). At high T cell doses infused, there could be sufficient numbers of CD28-/- T cells available to cause GVHD. Although CD28-/- T cells may respond to peptide and alloantigen, the response is not sustained, and therefore full expansion may not be achieved (33). The relative importance of CD28:B7 costimulation on GVHD induction will most likely depend upon the number and subset of T cells infused, threshold number of activated T cells required to induce GVHD, and rapidity and extent of GVHD-induced lethality in the control group.
In recipients of wild-type T cells, anti-CTLA-4 mAb administration led to 2–3-fold increase in the number of TDL T cells, especially CD4+ T cells, similar to results in a CD4+ T cell transgenic system (10). The preferential and CD28-dependent expansion of CD4+ T cells in response to blockade of CTLA-4:B7 interaction by anti-CTLA-4 mAb in our experiments has been observed in CTLA-4-/- mice that develop a lymphoproliferative disorder initiated by CTLA-4-/- CD4+ T cells (34). These data also are consistent with those of Abbas and colleagues who have shown that anti-CTLA-4 mAb block the induction of tolerance in vivo (14). The increase in TDL T cell number in anti-CTLA-4 mAb-treated BMT recipients was CD28 dependent. Wild-type and CD28-/- TDL cells generated CTLs capable of lysing host alloantigen, the magnitude of which was at least as high when using CD28-/- as was observed with wild-type TDL cells, consistent with other investigators (30, 31, 35) and our own data on granzyme B mRNA expression in CD28-/- TDL T cells. We interpret the data of Anasetti, who noted a marked reduction in the anti-host-reactive proliferative and CTL capacity of posttransplant spleen cells obtained from unirradiated F1 recipients of CD28-/- donor spleen cells to be reflective of the compromised alloengraftment capacity of CD28-/- T cells in that setting (32). When incubated with host-type stimulator cells, wild-type and CD28-/- TDL had a peak proliferative response on day 1 of culture, indicative of an in vivo priming against host alloantigens, the magnitude of which was greater with wild-type TDL. Anti-CTLA-4 mAb in vivo did not increase the magnitude of the proliferative response of wild-type or CD28-/- donor TDL T cells obtained at the height of peak in vivo alloresponsiveness, although the total number of donor TDL T cells was significantly increased. Even though the function of donor TDL T cells was not affected by mAb infusion, the net effect of anti-CTLA-4 mAb administration in wild-type recipients is to increase the number of donor TDL T cells with anti-host reactivity.
Our data indicate that the majority of anti-CTLA-4 mAb effects was CD28 dependent. Similar to our observations that CTLA-4:B7 interaction did not have a positive regulatory role of T cell proliferation, Green et al. showed that the in vitro alloresponses of CD28-/- T cells were not inhibitable by the addition of CTLA4-Ig fusion protein to the MLR culture (35). In contrast, others have shown that CTLA-4:B7 interaction can have a negative regulatory role on the capacity of CD28-/- recipients to respond to tumor Ags or alloantigen-bearing cardiac grafts (16, 36). The apparent conflicting results between these studies and our data may be related to the type of T cell, APC, Ag, murine strain, or exact type of immune response being measured, as well as the requirement for CD28:B7 interaction in that response. Nonetheless, our data clearly indicate that, under some conditions, the negative regulatory role of CTLA-4:B7 interaction is CD28 dependent.
An interesting observation in the present study is the finding that the percentage of TDL cells expressing Th2 cytokine mRNA was increased in recipients receiving allogeneic T cells and anti-CTLA-4 mAb infusion. Th2 skewing was independent of the presence of CD28. A similar skewing toward a Th2 cytokine phenotype was observed when CD4+CTLA-4-/- TCR transgenic T cells were reprimed with Ag in vitro (A.H.S., unpublished observations). Consistent with our results, CD28 costimulation has been shown to promote Th2 cytokine production (37). Recent data from our group have shown that GVHD lethality can be significantly reduced in heavily irradiated B10.BR recipients of allogeneic donor T cells from B6 Th2-defective (IL-4-deficient) donors (38), suggesting that the accelerated GVHD responses observed in anti-CTLA-4 mAb-treated recipients were due to Th2 skewing. However, the effect of Th1 or Th2 cytokine-producing donor T cells in regulating GVHD responses is most likely dependent upon the particular transplantation setting analyzed. For example, other data from our group have shown that T cells from IL-10-deficient donors cause an increase in GVHD lethality when infused into sublethally irradiated recipients (39). Other investigators have shown that CD4+ and CD8+ Th2 cells can be associated with a lower risk for GVH and less potent GVL than T cells with a Th1 phenotype (40, 41, 42). Thus, we do not know whether the anti-CTLA-4-facilitated induction of Th2 cells contributed to the observed increases in the GVH and GVL effects of wild-type T cells. Nonetheless, collectively, these data indicate that CTLA-4:B7 interaction can regulate Th2 differentiation.
We have shown that anti-CTLA-4 mAb administration had a profound effect on stimulating the GVL response of delayed splenocyte infusion in recipients challenged with MHC class I+II- AML cells, consistent with data from others that have shown that anti-CTLA-4 mAb administration stimulates a potent anti-tumor response in naive mice (43, 44). Because GVHD mortality is known to be lower when donor T cells are given later as contrasted to earlier post-BMT (21, 45), the later post-BMT infusion of anti-CTLA-4 mAb may be more tolerable in terms of GVH side effects than when given early post-BMT. The fact that anti-CTLA-4 mAb infusion did significantly increase GVL without GVHD in this setting does not indicate that GVL and GVHD are separable events. For example, if fewer donor T cells are required to mediate GVL than GVHD lethality, then the increase in donor T cell expansion or function induced by mAb infusion may be of a sufficient magnitude to support a potent GVL effect without an obvious increase in GVHD risk. In our GVL system, anti-B7 mAb infusion eliminates, while transduction of the tumor line with B7-1 markedly increases the GVL effect (21). Because AML B7-1 transduction is cumbersome and the GVH response of DLI was not significantly augmented by anti-CTLA-4 mAb infusion, the inclusion of anti-CTLA-4 mAb along with delayed lymphocyte infusion could represent a new approach to increasing the GVL efficacy in patients relapsing post-BMT.
In summary, our data would indicate that, for GVHD prevention, competitive antagonists of CD28:B7 interaction, which preserve CTLA-4:B7 binding, would be preferable to inhibitors of both pathways. Such an approach could inhibit GVHD generation without adversely affecting the engraftment of TCD BM. Because GVHD was observed when high numbers of CD28-/- CD4+ and CD8+ T cells were infused together, the coblockade of additional pathways such as the CD40 ligand:CD40 costimulatory pathway (29, 46) or an adhesion pathway (19) will be necessary to further reduce GVHD lethality. Anti-CTLA-4 mAb infusion was a potent means of augmenting the GVL effect of donor splenocytes given later post-BMT at a time period when the risks of graft rejection and GVHD are lower. The inclusion of anti-CTLA-4 mAb with DLI in patients that relapse post-BMT warrants investigation. The enhanced GVHD, graft rejection, and GVL responses in anti-CTLA-4 mAb-treated recipients are consistent with the hypothesis that CTLA-4:B7 interaction results in the delivery of a negative signal (5, 6, 7, 11, 12, 13), although Linsley and Liu have proposed that CTLA-4:B7 delivers a positive signal to the T cell (4, 8, 15). Whatever the true nature of the CTLA-4:B7 signaling of T cells, anti-CTLA-4 mAb could be exploited to have a beneficial effect on post-BMT outcome. Together, the data presented in this work have significant potential implications to modifying the CD28/CTLA-4:B7 pathways for humans undergoing BMT.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce R. Blazar, University of Minnesota Hospital, Box 109, Mayo Building, 420 S.E. Delaware Street, Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: CTLA, cytolytic T lymphocyte-associated Ag; BM, bone marrow; BMT, BM transplantation; DLI, delayed donor lymphocyte infusion; GVH, graft versus host; GVHD, GVH disease; GVL, graft versus leukemia; Gy, Gray; LN, lymph node; TBI, total body irradiation; TCD, T cell-depleted; TDL, thoracic duct lymphocytes.
Received for publication December 2, 1998. Accepted for publication March 11, 1999.
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and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Clin. Invest. 102:1742.[Medline]
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