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*
University of Minnesota Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, MN 55455;
Department of Opthalmology, Louisiana State University Medical Center, New Orleans, LA 70112;
Immunomodulation Research Center, University of Ulsan, Ulsan, Korea; and
Immunex, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal
number of donor splenocytes given later post bone marrow
transplantation by bolstering allogeneic responses resulting in
leukemia elimination. In summary, 4-1BB ligation is a potent regulator
of CD4+ and CD8+ T cell-mediated allogeneic
responses in vivo. Modifying the ligation of 4-1BB represents a new
approach to altering the graft-vs-host disease and graft-vs-leukemia
effects of allogeneic T cells post bone marrow
transplantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the expression of 4-1BB on both CD4+ and CD8+ T cells, the majority of data available to date has suggested that CD8+ T cells are affected to a greater extent by agonistic anti-4-1BB mAbs than CD4+ T cells (16, 17, 18, 19). In vivo CTL generation against both alloantigen (17) and tumor Ag-bearing targets (20) can be enhanced by the administration of a mitogenic anti-4-1BB mAb infusion. Results obtained with anti-4-1BB mAb infusion into tumor-bearing mice indicated that both CD4+ and CD8+ T cells were required for an optimal anti-tumor immune response, indicating that 4-1BB receptor signaling augmented T helper support of CD8+ CTL responses (20).
This study was conducted to determine the effect of 4-1BB receptor/4-1BBL interaction on regulating alloresponses in vivo. Therefore, we analyzed three types of clinically relevant in vivo alloresponses: graft-vs-host disease (GVHD), allogeneic bone marrow (BM) graft rejection, and graft-vs-leukemia (GVL). For this purpose, we used mice with a deletion in either 4-1BB or 4-1BBL and a mitogenic anti-4-1BB mAb. GVHD is caused by alloantigen-activated donor T cells that expand in vivo, migrate into GVHD target organs, and cause tissue destruction (reviewed in Refs. 21 and 22). In situations in which both CD4+ and CD8+ T cells can recognize host alloantigenic disparities, CD8+ T cells with CTL activity are thought to be driven to expand by CD4+ T cells that provide helper cytokines, which support CD8+ T cell growth. BM graft rejection occurs as a result of a host anti-donor reaction in which host T cells recognize alloantigens present on donor BM cells. GVL involves the recognition of leukemia-associated Ags by donor-immune effector cells. In many instances, donor T cells expand and mediate the elimination of leukemia cells, which express host type alloantigens. CD4+ or CD8+ alloreactive T cells are involved in each of these three immune responses that are critical to determining the long-term outcome of BM transplantation (BMT) recipients. Because CD28/B7 interactions are potent T cell costimulatory molecule pathways that are required for optimal GVHD (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) and GVL responses (34), we further sought to determine whether the effects of anti-4-1BB mAb were dependent upon amplification of CD28/B7 signals. Our data indicate that 4-1BB/4-1BBL interactions regulate both CD4+ and CD8+ T cell-mediated alloresponses in vivo. These data have potentially important clinical ramifications.
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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). B6-CD45.1 congenic mice were purchased from the National Institutes of Health (Bethesda, MD). B6 and BALB/c (H2d) 4-1BB receptor deletional (-/-) mice were generated as described (35), backcrossed nine generations, and bred at the University of Minnesota. 4-1BBL-/- mice were generated as described (36) and backcrossed for five generations to B6. Mice were bred and housed in a specific pathogen-free facility in microisolator cages. Donors and recipients were used at 8–10 wk of age.
Monoclonal Abs preparation
Anti-4-1BB mAb, clone 1AH2, rat IgG1 (6), was
generated as ascites fluid and purified by ammonium sulfate
precipitation followed by dialysis. This anti-4-1BB mAb was found
to be mitogenic based on studies in which in vitro proliferation
consistently was enhanced when CD4+ T cells were
exposed to microspheres coated with suboptimal amounts of
anti-CD3
along with either anti-4-1BB or irrelevant control
mAb (data not shown). Control rat IgG was purchased from Rockland
Laboratories (Gilbertsville, PA). mAb injections were given i.p.
according to doses and schedules described below.
GVHD generation
To determine the effect of 4-1BB/4-1BBL 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 (37). 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 6.0 Gy total body irradiation (TBI) on day 0 from a 137Cesium source at a dose rate of 85 cGy/min (29, 33). Four to six hours after TBI, sublethally irradiated bm12 recipients were given purified lymph node (LN) CD4+ T cells at doses of 0.3–3 x 105 per recipient. To measure CD8+ T cell GVHD lethality, bm1 recipients were injected with CD8+ T cells at doses of 1.0 or 3.0 x 105 per recipient. Recipients were given irrelevant or anti-4-1BB mAb (200 µg/dose) i.p. from days -1 to +5 then thrice weekly through day +21 or +28 post transfer, as indicated. To measure the effect of 4-1BBL deficiency on CD4+ or CD8+ T cell-mediated GVHD lethality, bm12 CD4+ T cells or bm1 CD8+ T cells were infused into sublethally irradiated B6 4-1BBL-/- mice.
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
David Sachs, Massachusetts General Hospital, Charlestown, MA) or
anti-CD4 (hybridoma GK1.5, rat IgG2b; provided by Frank Fitch,
University of Chicago, 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 (29, 33). For all GVHD and engraftment systems, mice were monitored
daily for survival and clinical appearance and weighed twice
weekly.
Because irradiation-induced tissue injury may influence the requirement for costimulation to drive donor T cell expansion, we used two different types of strategies. In the first, mice were lethally irradiated with 8.0 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-Thy1.2 (clone 30-H-12; provided by David Sachs) plus rabbit complement treatment (23, 27). To measure CD4+ and CD8+ T cell GVHD responses, irradiated B10.BR recipients were infused i.v. with 8 x 106 TCD B6 BM along with 0, 5, or 15 x 106 splenocytes from B6 or 4-1BB-/- donors. To measure CD4+ T cell GVHD responses under conditions of heavy irradiation, bm12 recipients were lethally irradiated (800 cGy TBI) and then infused with 20 x 106 TCD BM along with 0 or 0.3 x 106 purified T cells from B6 or 4-1BB-/- or CD28-/- donors (33).
Thoracic duct cannulation
For thoracic duct lymphocyte (TDL) isolation, cannulae were inserted into the thoracic duct of recipients at the time of peak proliferation (day 6) post-BMT. TDL were collected over a period of 18 h (29, 33). Irrelevant or anti-4-1BB mAb was given at a dose of 200 µg daily i.p. from days -1 to +6 post-BMT.
GVL studies
Our GVL induction procedure has been described in detail (34). In brief, B6 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 or 20 x 106 BALB/c TCD BM cells on day 0. Some mice, as indicated, were administered splenocytes from B10.BR, BALB/c, or BALB/c 4-1BB-/- donors i.v. at the indicated doses on day 21 post-BMT. On day 28 post-BMT, mice were challenged with an MHC class I+ II- acute myeloid leukemia line, C1498 (34). C1498 (American Type Culture Collection, Manassas, VA), originally derived as a spontaneous tumor line from a B6 mouse, was grown in RPMI 1640 (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 were given via the i.v. route at various cell doses ranging from 2 x 104 to 2 x 106 cells/mouse. In some experiments, cohorts of mice were given either irrelevant mAb or anti-4-1BB mAb at 200 µg/dose beginning 1 day before splenocyte infusion or 1 day before tumor infusion in mice that do not receive splenocytes. mAb is continued daily for 1 wk and then two times per week for three additional weeks. All available animals were examined for evidence of GVHD and tumor by necropsy (34).
Engraftment studies
B6-CD45.1 mice were irradiated with 5.0 or 5.5 Gy TBI, as indicated, by x-ray on day -1 and then given bm1 or bm12 TCD BM (5 x 106) cells (33, 38). Recipients were given anti-4-1BB or irrelevant mAb (200 µg/dose) i.p. daily from days -1 to +6 then twice weekly through day 14. Peripheral blood cells were phenotyped 6 wk post-BMT.
Flow cytometry
The T cell, B cell, and granulocyte/macrophage constituency of
splenocytes, peripheral blood cells, or TDL effluent was 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 recipients 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 U. Hammerling (Memorial Sloan
Kettering Cancer Center, New York, NY). 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
SA-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 assessment
For quantifying MLR responses, CD4+ LN T
cells were mixed with irradiated (30 Gy) TCD splenocyte stimulators
from host strain mice as described (33). Cells were
suspended in MLR medium consisting of Dulbecco’s minimal essential
medium (BioWhittaker, Walkersville, MD), 10% FCS (HyClone), 2-ME
(5 x 10-5 M) (Sigma, St. Louis, MO), 10 mM
HEPES buffer, 1 mM sodium pyruvate (Life Technologies), and amino acid
supplements (1.5 mM L-glutamine, L-arginine,
and L-asparagine) (Sigma), antibiotics (penicillin, 100
U/ml; streptomycin, 100 µg/ml; Sigma). For microtiter well analysis,
105 cell responders and 105
irradiated stimulators were plated in replicates of six into 96-well
round-bottom (Costar) plates and placed at 37°C and 10%
CO2 for 2–8 days. In some wells, as indicated,
anti-4-1BB mAb was added at a final concentration of 50 µg/ml.
Bulk cultures were established with 0.5 x
106 responders and 0.5 x
106 irradiated stimulators/ml and cultured under
the same conditions as used for microtiter wells. Supernatant was
collected from bulk cultures on the days indicated for ELISA analysis
of cytokines. Cell recovery was quantified on day 8. Flow cytometry was
performed at the end of the culture to analyze responder cells for
expression of activation Ags (CD25, CD80, CD86, CD137, CD62 ligand; all
were obtained from PharMingen), forward- and side-scatter properties,
and signs of early apoptosis (7-amino-actinomycin D). To measure
restimulation responses, day 8 bulk MLR cultures were extensively
washed free of mAb and plated at a concentration of 0.3 x
105/well along with
105/well of irradiated splenocyte stimulators.
Bulk cultures were established to obtain supernatants for cytokine
analysis using 0.15 x 106/ml responders and
0.5 x 106/ml irradiated stimulators.
Microtiter wells were pulsed with tritiated thymidine (1 µCi) for
18 h before harvesting and counted in the absence of scintillation
fluid on a beta plate reader. 
cpm were calculated by subtracting
the syngeneic proliferative response from the allogeneic proliferative
response.
The frequency of proliferating T lymphocyte precursor (pPTL) was quantified. Eight 3-fold serial dilutions of responder CD4+ T cells were plated at 30 replicates in 96-well round-bottom plates and incubated for 7 days with irradiated (30 Gy), non-TCD splenic stimulators in the absence of IL-2. Anti-4-1BB mAb was added to the primary LDA at a final concentration of 50 µg/ml. Wells were pulsed with tritiated thymidine for 18 h before harvesting. Wells were scored positive if their cpm exceeded the average cpm plus 3 SDs of the stimulators plated without responders. Using Poisson distribution statistics according to the method of Taswell and with the aid of a computer program, the likelihood of a single hit was confirmed and a frequency estimate was calculated.
For CTL analysis, CTL killing was assessed by the JAM assay (39). To generate targets, splenocytes were activated with Con A (2 µg/ml) for 3 days in MLR medium as described above. Con A-activated splenocyte target cells were labeled overnight with [3H]thymidine (4 µCi/ml) in 24-well plates containing 2 x 106 cells in a 2-ml volume. TDL effectors were spun at 500 rpm (Beckman TJ-6; Beckman Coulter, Fullerton, CA) for 5 min with targets (104 targets/well) at E:T ratios of 12.5:1–0.8:1 in the above described medium 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 (25).
Quantitation of cytokine levels by ELISA
Murine cytokine levels (IL-2, IL-4, IL-10, TNF-, IL-13,
IFN-
) in the supernatants of MLR cultures were quantitated by ELISA
(R&D Systems, Minneapolis, MN). Sensitivity of the assays was between 1
and 10 pg/ml for each assay. A standard curve using recombinant protein
was generated with each assay.
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
(40). Actuarial survival and relapse rates are shown.
Probability (p) values 
0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because 4-1BB is a known CD8+ T cell
survival factor (16), we first determined whether
anti-4-1BB mAb infusion would augment CD8+ T
cell-mediated GVH lethality. Sublethally irradiated bm1 recipients were
given a sublethal number of highly purified B6 MHC class I disparate
CD8+ T cells (105). Mice
that received a mitogenic anti-4-1BB mAb had a 75% lethality rate
that was significantly higher than the 0% observed in controls (Fig. 1
). Weight curves reflected a greater
degree of weight loss in the mAb-treated group, which was evident as
early as day 7 and reached a maximum difference on day 19 post transfer
(data not shown). The 75% lethality rate in mAb-treated recipients
given 105 T cells was not statistically different
from the 90% lethality rate observed in controls receiving a 3-fold
higher CD8+ T cell number (Fig. 1).
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A). In contrast to the 0%
lethality by day 99 post-BMT in the irrelevant mAb-treated control
group, 100% of the anti-4-1BB mAb-treated group died by day 10
post-BMT. To confirm that 4-1BB/4-1BBL interaction was essential for
optimal CD4+ T cell-mediated lethality, we
performed experiments in which donor CD4+ T cells
obtained from B6 4-1BB-/- mice were infused
into sublethally irradiated bm12 recipients. Data pooled for two
replicate experiments with similar results are shown in Fig. 2B. As compared with recipients of B6 wild-type
CD4+ T cells, recipients of
4-1BB-/- CD4+ T cells had
a >3-fold reduction in GVH lethality based on the observation that
mice receiving 105
4-1BB-/- CD4+ T cells had
a 45% lethality rate vs a 94% lethality rate in recipients of as few
as 3 x 104 wild-type cells.
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C). As observed
with 4-1BB-/- donor T cells, there was a
>3-fold reduction in GVH lethality in
4-1BBL-/- recipients vs controls, which was
most evident by comparing the survival curves of 4-1BBL recipients
given 3 x 105 T cells as compared with
wild-type control recipients given 105 T cells.
Under these adoptive transfer conditions, recipients experience
multiorgan system GVH-induced damage as well as BM destruction due to
the reaction of donor T cells against host hemopoietic progenitor cells
(29, 33). A convenient measurement of GVH-induced injury
is the quantification of peripheral blood hematocrit values, which can
be sequentially analyzed without sacrificing recipients (29, 33). As compared with wild-type controls, day 14 hematocrit
values obtained from 4-1BBL-/- recipients of
3 x 105 CD4+ T cells
indicated a reduction in GVH-mediated BM destruction (17 ± 6 vs
23 ± 5%; p = 0.067). We conclude that 4-1BB
regulates CD4+ T cell-mediated GVH responses to
approximately the same extent as CD8+ T cell
responses in sublethally irradiated recipients.
To directly examine the effect of anti-4-1BB mAb on
CD4+ T cell responses, in vitro studies were
performed using B6 CD4+ T cells and TCD,
irradiated bm12 stimulators. MLR results indicated that anti-4-1BB
mAb did not increase proliferation or cell recovery as compared with
controls (data not shown). Activation Ag expression (CD25, CD69, CD80,
CD86, CD62 ligand) on CD4+ T cells was similar
for each one in the two groups (data not shown). Restimulation of
washed control or anti-4-1BB mAb-treated primary MLR cultures did
not reveal any differences upon repriming of CD4+
T cells previously exposed to anti-4-1BB mAb vs control cultures
(data not shown). Supernatants obtained from primary MLR cultures (days
2, 4, 6, and 8) and secondary MLR cultures (days 1, 3, and 5) revealed
similar levels of Th1 (IL-2, IFN-, TNF-) cytokines (data not
shown). For the most part, Th2 (IL-4, IL-10, IL-13) were comparable
between the two groups although at some time periods, in secondary MLR
cultures, supernatants from anti-4-1BB mAb-treated cells appeared
to have higher Th2 (IL-10) concentrations detectable (data not shown).
The lack of effect of anti-4-1BB mAb on CD4+
T cell-proliferative responses is consistent with results obtained in a
proliferating T lymphocyte assay in which the precursor frequency of B6
CD4+ T cells responding to bm12 allogeneic
stimulator cells was unchanged by anti-4-1BB mAb (1:6788 vs 1:7202,
respectively).
To determine whether anti-4-1BB mAb infusion would help prime
CD4+ alloreactive T cells in vivo, lethally
irradiated irrelevant or anti-4-1BB mAb-treated bm12 recipients
were given B6 TCD BM plus supplemental B6 CD4+ T
cells (106/recipient). On day 6 post-BMT, TDL
were obtained and analyzed for in vitro priming to TCD bm12 stimulators
in an MLR culture. Four mice per group were individually studied. As
observed with the in vitro MLR culture using purified
CD4+ LN T cells, TDL exposed to anti-4-1BB
mAb in vivo did not have a significantly greater proliferative response
to MHC class II-disparate host-strain stimulators (Fig. 3). Cytokine analysis of MLR culture
supernatants revealed comparable levels of Th1 cytokines in these two
groups, although as observed with in vitro alloprimed
CD4+ T cells, some Th2 cytokines (IL-4, IL-13)
are increased in cultures in which CD4+ T cells
have been previously exposed to anti-4-1BB mAb (Table I). The lack of effect of anti-4-1BB
mAb on CD4+ T cell-proliferative responses in
these assays is consistent with results obtained for in vivo T cell
expansion data as assessed by thoracic duct cannulation studies (see
below). We hypothesize that the acceleration in
CD4+ T cell-mediated GVHD responses we have
observed with anti-4-1BB mAb infusion in vivo is not due to support
of T cell proliferation or expansion, although we cannot exclude an
effect of anti-4-1BB mAb on functioning as a survival factor by
supporting late T cell expansion in vivo.
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). In
this strain combination, both CD4+ and
CD8+ T cells are required for optimal GVH-induced
lethality (26). Recipients of
4-1BB-/- splenocytes at two different doses had
significantly higher survival rates. Moreover, recipients of a 3-fold
higher number of 4-1BB-/- vs wild-type
splenocytes (15 x 106 vs 5 x
106, respectively) had a statistical trend
(p = 0.079) toward a higher survival rate,
consistent with a 
3-fold reduction of GVHD-induced lethality. At
both splenocyte doses, weight curves were reflective of survival
outcome (data not shown). Thus, 4-1BB/4-1BBL interactions are an
important regulator of CD4+ and
CD8+ T cell-mediated GVHD in heavily irradiated
recipients.
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We next sought to obtain information as to the mechanism by which
GVHD was regulated by 4-1BB/4-1BBL in lethally irradiated B10.BR
recipients of B6 donor splenocytes. To ensure adequate numbers of cells
to be available, we infused wild-type rather than
4-1BB-/- splenocytes (containing a total of
3.6 x 106 CD4+ T
cells and 2.3 x 106
CD8+ T cells) as a source of donor cells and then
treated mice with either anti-4-1BB or irrelevant mAb from days -1
to +6 post-BMT. At that time, thoracic duct cannulation was performed
as a means of analyzing alloreactive donor T cells early post-BMT. Day
6 post-BMT is the time of maximal in vivo donor T cell expansion in
response to the allogeneic recipient (32), paralleling the
results of in vitro MLR assays. The total number of donor T cells
(n = 4/group) produced per day (mean ± 1 SD
= 46 ± 8 vs 34 ± 6 x 106,
irrelevant vs anti-4-1BB mAb, respectively; p =
0.16), the total number of CD4+ T cells (12
± 3 vs 12 ± 3 x 106, respectively;
p = 0.84) and the total number of
CD8+ T cells (35 ± 7 vs 24 ± 4
x 106, respectively; p = 0.10)
were not significantly different between the groups. The lack of effect
of anti-4-1BB mAb on donor T cell expansion was reproduced in a
second experiment (data not shown). CTL killing against irradiated
splenic host-strain stimulators at E:T ratios of 6.25:1 was
significantly increased in the anti-4-1BB mAb-treated group despite
the fact that there was a lower proportion of
CD8+ TDL cells compared with the controls (data
not shown). However, with E:T ratios of 12.5:1, CTL killing was
comparable in both groups (data not shown). These data would indicate
that although anti-4-1BB mAb does not augment to any great extent
either CD4+ or CD8+ T cell
proliferation in vivo under these conditions, donor
anti-host-reactive CTLs are modestly increased by anti-4-1BB
mAb treatment.
The accelerated GVHD lethality mediated by anti-4-1BB mAb-stimulated CD4+ T cells is partially CD28 independent
Ligation of 4-1BB can provide a T cell-costimulatory signal to
either CD28+ or CD28- T cells (e.g., Refs.
9, 42). For CD28+ T cells, 4-1BB signaling
can lower the threshold of CD28 signaling required to sustain T cell
proliferation and IL-2 production. Because CD28/B7 and 4-1BB/4-1BBL can
act independently or synergistically in inducing different types of T
cell responses, we asked whether the augmented
CD4+ T cell-mediated GVHD responses observed with
anti-4-1BB mAb could function in a CD28-independent fashion in
vivo. Lethally irradiated bm12 were reconstituted with B6 TCD BM
supplemented with purified CD4+ T cells (1 or
3 x 106) from
CD28-/- donors. Recipients of
106
CD4+CD28-/- T cells were
given either irrelevant or anti-4-1BB mAb. Anti-4-1BB mAb
accelerated GVHD lethality such that 100% of recipients of
anti-4-1BB mAb vs 60% of control recipients died with GVHD in the
first 2.5 mo post-BMT (p = 0.079). Recipients
of 3 x 106
CD4+CD28-/- T cells all
died by day 12 post-BMT. Thus, the degree of GVHD acceleration was
<3-fold (3 x 106 T cells vs
106 T cells and anti-4-1BB mAb,
p = 0.068). Because recipients of 0.3 x
106
CD4+CD28+ T cells had no
lethality, and those that received anti-4-1BB mAb all died within
10 days post-BMT (Fig. 2A), these data collectively indicate
that 4-1BB mAb-induced GVHD can occur in a CD28-independent fashion
although CD28/B7 interaction increases the function of 4-1BB mAb in
inducing GVHD.
4-1BB/4-1BBL interaction augments allogeneic BM rejection mediated by either CD4+ or CD8+ donor T cells
The data presented above clearly indicate that anti-4-1BB mAb
accelerates GVHD induction by either CD8+ or
CD4+ donor T cells that encounter MHC class I or
II Ags, which are distributed throughout the host microenvironment. To
determine whether 4-1BB signaling regulates host
CD4+ or CD8+ T cells that
can recognize MHC Ags present on donor BM cells, we tested
anti-4-1BB mAb administration for effects on alloengraftment.
Anti-4-1BB mAb administration augmented host anti-donor T
cell-mediated graft rejection by either T cell subtype. In a host
CD8+ T cell-mediated graft rejection system
(38), as compared with controls, mice that received MHC
class I only disparate cells and anti-4-1BB mAb had a significantly
higher proportion of host cells present in the periphery (10 ±
0% vs 40 ± 9%, respectively; p < 0.005) early
post-BMT (6 wk post-BMT) (data not shown). These differences persisted
when mice were analyzed later post-BMT (4 mo post-BMT) and were noted
to involve all lineages examined (Table II). In a host anti-donor
CD4+ T cell-mediated graft rejection system
(38), as compared with controls, mice that received MHC
class II only disparate cells and anti-4-1BB mAb had a
significantly higher proportion of host cells present in the periphery
(26 ± 2 vs 52 ± 2%, respectively; p <
0.005) at 6 wk post-BMT (data not shown). As observed for the
CD8+ T cell-mediated graft rejection system,
additional phenotyping analysis at 111 days post-BMT revealed that
chimerism was stable and further indicated that anti-4-1BB
mAb-treated recipients had significantly higher proportions of host
CD4+, CD8+,
CD19+, and Mac1+ cells,
reflective of an effect of mAb on facilitating rejection of allogeneic
donor stem cells (Table II). These data are consistent with the GVHD
results, demonstrating a role for both CD8+ and,
interestingly, CD4+ T cells in regulating
alloresponses in vivo.
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For patients that relapse post-BMT, DLI are used as a means of
achieving remission by providing a potent anti-leukemia effect via
recognition of alloantigens present on leukemia cells. Anti-4-1BB mAb
augments the GVHD effect of donor spleen cells infused into lethally
irradiated allogeneic recipients on day 0 of BMT. Because the GVHD
response is markedly diminished when donor spleen cells are given later
post-BMT (e.g., Refs. 34, 43), we performed studies to
determine whether anti-4-1BB mAb would facilitate a GVL effect
without augmenting GVHD lethality. B6 recipients were lethally
irradiated and reconstituted with B6 donor TCD BM. At 3 wk post-BMT
donor spleen cells were infused. One week later mice were challenged
with B6 AML cells. A low donor splenocyte dose (5 x
106) was chosen to minimize GVH potential, which
would be achieved in the irrelevant mAb-treated control groups.
Control-treated recipients given DLI all died within 3 mo post-BMT
(Fig. 5A) with AML (Fig. 5B). In contrast,
anti-4-1BB mAb-treated recipients of DLI had a 70% survival rate
at >6 mo post-BMT (p = 0.0054 vs irrelevant
mAb controls) (Fig. 5A). Ninety to 100 percent of recipients
given AML cells with or without DLI died with AML occurrence unless
anti-4-1BB mAb also was given (Fig. 5B). In the latter
instance, AML was found in only 30% of recipients
(p = 0.0012 vs irrelevant mAb control). Thus,
the combination of low dose DLI and anti-4-1BB mAb resulted in >2
log10 reduction in AML-associated lethality.
|
, A
and B). Recipients given the same donor splenocyte and high
AML dose inocula along with anti-4-1BB mAb had only a 20%
long-term survival rate (p = 0.014 vs
irrelevant mAb-treated controls). Anti-4-1BB-treated mice given donor
splenocytes and a 10-fold lower AML cell dose also had a significantly
lower survival rate compared with irrelevant mAb-treated controls
(p = 0.0012) (data not shown). The higher
mortality rate of anti-4-1BB mAb-treated recipients was not due to
a higher AML burden. None of the DLI-treated recipients of
anti-4-1BB mAb had evidence of AML when examined at autopsy as
compared with 30% of recipients of irrelevant mAb
(p = 0.11), indicating that anti-4-1BB mAb
stimulated a GVL effect albeit at the expense of GVHD-induced lethality
(Fig. 6B). Examination of the weight curves is supportive of
the latter conclusion. The effect of anti-4-1BB mAb on augmenting
DLI-mediated GVHD was also observed in B6 recipients given AML cells
and high doses of donor splenocytes (25 x
106) (data not shown). To avoid any contribution
of AML cells to the mortality process observed with DLI and
anti-4-1BB mAb, studies were performed in which high dose DLI
(50 x 106 splenocytes) was administered to
recipients not challenged with AML cells (Fig. 6C).
Recipients given anti-4-1BB mAb had a significantly lower actuarial
survival rate vs irrelevant mAb controls (0 vs 60%, respectively;
p = 0.00018), indicating that anti-4-1BB mAb
accelerated DLI-GVHD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that 4-1BB/4-1BBL interaction affects alloreactive CD4+ T cell responses by several different approaches that used anti-4-1BB mAb, 4-1BB receptor -/- donor CD4+ T cells, or 4-1BBL-/- recipients of donor CD4+ T cells. Both CD8+ and CD4+ T cell-mediated GVHD responses were affected by 4-1BB/4-1BB ligation. Based upon T cell dose titration studies, we have shown that CD4+ T cells were affected to approximately the same extent as CD8+ T cells. The generation of donor CD4+ T cells with anti-host CTL capacity that was facilitated by anti-4-1BB mAb administration was directly demonstrated by isolation of T cells obtained from allogeneic recipients. Our previous studies demonstrating that perforin-deficient donor T cells have a reduced GVHD lethality capacity in vivo indicate that CTL generation is important for T cell-mediated GVHD (44). Interestingly, in this study, we found that neither CD4+ nor CD8+ T cell expansion, as measured by thoracic duct cannulation, in vivo was increased by anti-4-1BB mAb infusion. Thus, our results would suggest that other studies demonstrating that anti-4-1BB mAb is more potent in stimulating CD8+ vs CD4+ T cells may be due to the particular anti-4-1BB mAb clone used in their studies (17, 18). Our studies extend those of Shuford and colleagues (17), who have shown that anti-4-1BB mAb infusion supported alloreactive CD8+ CTL generation in nonirradiated F1 recipients of parental spleen cells, which was associated with host splenic involution and CD8+ but not CD4+ T cell expansion.
Previous studies have shown using a panel of mAbs that the most potent
anti-4-1BB mitogenic mAbs tested were more effective in stimulating
CD8+ vs CD4+ T cells under
conditions of suboptimal CD3 stimulation. These investigators showed
that there were quantitative and qualitative differences in tyrosine
phosphorylation patterns between anti-4-1BB mAb-stimulated
CD8+ vs CD4+ T cells
(17). Anti-4-1BB mAb administration has been found to
rescue the superantigen-mediated deletion of CD8+
T cells to a greater degree than CD4+ T cells
(16). Tan et al., using 4-1BBL-/-
mice, have shown that 4-1BBL-/- mice had 2- to
3-fold lower CD8+ T cell expansion rates in
response to lymphocytic choriomeningitis virus (LCMV) without effects
on virus-specific CD4+ T helper responses
indicating a preferential effect of 4-1BBL on supporting
CD8+ vs CD4+ T cell
expansion in vivo (18). Therefore, we had anticipated that
anti-4-1BB mAb might have had pronounced effects on
CD8+ vs CD4+ T
cell-mediated GVHD. However, several studies have shown that
4-1BB/4-1BBL binding stimulates CD4+ T cell
responses (12, 16, 17, 18, 42, 45, 46). Moreover, the initial
characterization of 4-1BB receptor by Kwon and Weissman demonstrated
mRNA to be present in CTL and T helper cell lines (5) and
the 4-1BB receptor is expressed on both activated
CD4+ and CD8+ T cells
(6). Additional evidence that CD4+ T
cells can be affected by 4-1BB can be derived from studies by Mittler
and colleagues, who have shown that anti-4-1BB mAb suppresses in
vivo humoral responses to T cell-dependent Ags by a process that did
not directly inhibit either CD8+ T cells or B
cells but did presumably inhibit CD4+ T cells
(47). Optimal anti-4-1BB mAb-induced anti-tumor
immune responses were dependent on the presence of both
CD8+ and CD4+ T cells,
indicating that CD4+ T cells were enhanced by
anti-4-1BB mAb treatment (20). Other studies have
reported that 4-1BBL can augment in vitro CD4+ T
cell proliferation, cell cycle progression, and IL-2 secretion, and
suppress cell death (46). Our data extend these
observations by directly demonstrating that naive
CD4+ T cells are regulated by 4-1BB/4-1BBL
interactions in vivo under clinically relevant conditions. MLR studies
with the particular anti-4-1BB mAb clone (1AH12) used in our
studies did not reveal any evidence of enhancement of alloresponses in
vitro although with suboptimal (but not optimal) engagement of the
CD3 chain we were able to demonstrate that the anti-4-1BB mAb we
used was indeed mitogenic. The lack of enhancement of proliferative
responses to alloantigen would suggest that the mechanism(s) involved
in the augmentation of GVHD responses in vivo by the binding of 4-1BB
to its ligand is not due to an increase in the early expansion phase of
alloreactive CD4+ T cells that may already be
maximally driven by the recognition of host alloantigen-bearing cells.
Rather, we favor the possibility that 4-1BB/4-1BBL interaction
functions either to increase donor anti-host CTL-mediated tissue
injury or to sustain CD4+ T cells past the point
of peak in vivo expansion. 4-1BB costimulation has been shown to
preferentially support Th1 cell cytokine production while suppressing
Th2 cytokine production (45). Others have shown that the
enhancement of Tc1 cytokine production is observed only with
CD8+ T cells (17), whereas Th1 or
Th2 cytokine production in Th1 or Th2 clones was not affected by 4-1BB
binding (46). Our data with alloactivated
CD4+ T cells demonstrate that neither Th1 nor Th2
cytokine production is substantially affected by anti-4-1BB mAb
exposure, consistent with data in CD4+ T cell
clones (47). Therefore, we do not favor as an explanation
that the augmentation of CD4+ T cell-mediated
GVHD by 4-1BB ligation is due to a shift in the cytokine profile to Th1
cytokines. Regardless of the mechanism(s) responsible for GVHD
augmentation, the fact that both CD4+ and
CD8+ donor T cells are regulated by 4-1BB/4-1BBL
interactions have significant implications for the clinical GVHD
setting as both T cell types are present in the donor BM inoculum.
Results by our group and others (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) have shown that optimal GVHD generation requires CD28/B7 interaction. These studies demonstrate that anti-4-1BB mAb-induced CD4+ T cell-mediated GVHD acceleration can occur albeit to a lesser extent with CD28-/- donor T cells. These data would indicate that GVHD inhibition would be more effectively achieved by blocking both CD28/B7 and 4-1BB/4-1BBL pathways. Our findings that 4-1BB mAb-induced GVHD acceleration was less potent when infusing CD28-/- vs wild-type T cells are consistent with investigators who have shown that 4-1BB receptor signaling can enhance proliferation of activated T cells that are dependent on T cell costimulation but are incapable of being costimulated through the CD28/CTLA4:B7 pathway (9, 12, 14, 36, 42, 45).
Although Melero et al. have shown that anti-4-1BB mAb was a potent inducer of CTL generation resulting in the rejection of established, poorly immunogenic tumors (20), we were unable to observe any benefit of anti-4-1BB mAb in bolstering an immune response to systemically administered AML cells unless alloreactive DLI were given. Our previous studies have shown that CD8+ T cells are the cytolytic effectors for the MHC class I+ II- AML cells used in this study. CD4+ T cells may provide help for this response. The fact that anti-4-1BB mAb treatment in either non-BMT mice or in allogeneic BMT recipients not given DLI failed to result in an observable anti-AML effect may be due to the fact that the immune response to AML cells under these conditions is weak and, in the absence of priming, anti-4-1BB mAb may not be expected to further increase the response. Alternatively, it is possible that the differences of our results as compared with Merlero et al. are related to the particular anti-4-1BB mAb clone used. Nonetheless, because anti-4-1BB mAb-treated recipients of allogeneic DLI can mount a vigorous anti-AML effect, it is likely that anti-4-1BB mAb augments alloreactivity of DLI, which is capable of eliminating AML cells via recognition of alloantigens present on the AML cells. The GVH response of DLI may be particularly amenable to an augmented GVL response under the cover of anti-4-1BB mAb because alloreactive T cells can up-regulate 4-1BB receptor expression, thereby further amplifying the mitogenic effect of anti-4-1BB mAb. Anti-4-1BB mAb infusion may be particularly beneficial in settings in which the baseline alloreactive response is suboptimal in terms of eliminating AML cells in vivo and may be far less critical if alloreactive T cells are potent mediators of GVL responses. For example, we have found that B6 recipients of BALB/c 4-1BB-/- DLI are still capable of 2 log10 elimination of AML cells in vivo (our unpublished observations). Conversely, anti-4-1BB mAb may be inadequate in supporting a GVL effect in situations in which alloreactivity is profoundly suboptimal as is the case with allogeneic BMT recipients of TCD donor grafts that are not given DLI. Because anti-4-1BB mAb facilitates the alloreactive potential of DLI, recipients receiving both DLI and anti-4-1BB mAb would be at risk for greater GVHD-induced complications. Nonetheless, there are situations in which DLI are not especially efficacious in providing long-term disease-free survival for patients with leukemia relapsing post-BMT (e.g., acute lymphoblastic leukemia or AML) (48). In those patients, the increased risk of GVH-associated complications may be offset by the possibility of superior long-term survival due to the generation of a more potent GVL effect.
In summary, we have shown that 4-1BB/4-1BBL interactions regulate both the CD4+ and CD8+ T cell-mediated GVHD response of donor T cells given at the time of BMT or later post-BMT. In the latter instance, the GVL response of DLI could be sufficiently improved so as to translate into a superior long-term survival rate. The host anti-donor T cell-mediated BM allograft rejection mediated by both CD4+ and CD8+ T cells was shown to be dependent in part on 4-1BB/4-1BBL interactions. The fact that the 4-1BB receptor costimulatory pathway had such a pronounced effect on CD4+ T cell responses was somewhat unexpected. Furthermore, we have shown that the augmented responses of CD4+ T cells to anti-4-1BB mAb were due to effects on CTL generation and not due to either a preferential induction of Th1 or Th2 cytokine production or to increased proliferation. The increased in vivo GVHD responses of alloreactive CD4+ T cells exposed to anti-4-1BB mAb were only partly CD28 dependent. Thus, all three major allogeneic responses (GVHD; graft rejection; GVL), which are critical in determining the long-term outcome of allogeneic BMT, were affected by 4-1BB/4-1BBL interactions. Our data suggest that manipulation of the 4-1BB/4-1BBL costimulatory pathway alone or in conjunction with the CD28/CTLA4:B7 pathway could represent an important therapeutic modality for improving the outcome of allogeneic 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; MMC 109, 420 Southeast Delaware Street, Minneapolis, MN 55455.
3 Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; BM, bone marrow; BMT, BM transplantation; GVHD, graft-vs-host disease; GVL, graft-vs-leukemia; LN, lymph node; TBI, total body irradiation; TCD, T cell-depleted; TDL, thoracic duct lymphocyte(s).
Received for publication October 24, 2000. Accepted for publication December 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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