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Analysis of the Requirements for the
Induction of CD4 + T Cell Alloantigen
Hyporesponsiveness by Ex Vivo Anti-CD40
Ligand Antibody
Patricia A. Taylor, Angela Panoskaltsis-Mortari, Randolph J.
Noelle and Bruce R. Blazar
J Immunol 2000; 164:612-622; ;
doi: 10.4049/jimmunol.164.2.612
http://www.jimmunol.org/content/164/2/612
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Copyright © 2000 by The American Association of
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References
Analysis of the Requirements for the Induction of CD4ⴙ T
Cell Alloantigen Hyporesponsiveness by Ex Vivo Anti-CD40
Ligand Antibody1
Patricia A. Taylor,* Angela Panoskaltsis-Mortari,* Randolph J. Noelle,† and Bruce R. Blazar2*
B
one marrow transplantation (BMT)3 is being utilized as
the standard of care for the treatment of a number of
malignant and nonmalignant disorders of both hematologic and nonhematologic origin. Graft-vs-host disease (GVHD)
remains a major cause of morbidity and mortality after sibling,
matched unrelated, haploidentical, or mismatched bone marrow
transplantion (1). Although GVHD can be prevented by rigorous T
cell depletion ex vivo of the donor graft or prolonged, global immunosuppression in vivo in the recipient, these strategies have
associated risks of increased rates of leukemic relapse, infectious
complications, or graft failure (1). A strategy that would selectively target only that small fraction of donor T cells capable of
mediating GVHD but preserve those T cells that might mediate
antitumor, anti-infectious effects and promote engraftment would
be desirable. An in vitro blockade strategy that would not involve
global in vivo immune suppression or nonspecific multiorgan toxicity also would be advantageous.
Productive T cell activation and proliferation require two signaling events. The first signal is the engagement of the TCR with
*University of Minnesota Cancer Center and Department of Pediatrics, Division of
Bone Marrow Transplantation, Minneapolis, MN 55455; and †Department of Microbiology, Dartmouth Medical College, Hanover, NH 03756
Received for publication September 10, 1999. Accepted for publication October
27, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants RO1 AI34495, R37 HL-56067, and PO1 AI-35296.
2
Address correspondence and reprint requests to Dr. Bruce R. Blazar, Box 109 Mayo
Building, University of Minnesota Hospital, 420 SE Delaware Street, Minneapolis,
MN 55455. E-mail address: [email protected]
3
Abbreviations used in this paper: BMT, bone marrow transplantation; FSC, forward
scatter; GVHD, graft-vs-host disease; HCT, hematocrit; LDA, limiting dilution assay;
SSC, side scatter.
Copyright © 2000 by The American Association of Immunologists
the MHC-peptide ligand complex on the surface of the APC. Additional costimulatory signals are required for the full activation of
the intracellular signaling cascade, IL-2 production, and ultimate T
cell proliferation (2–7). Multiple pathways capable of costimulation have been described, including CD40L:CD40, CD28/CTLA4:
B7-1/B7-2, OX40:OX40L, 41BB:41BBL, CD30:CD30L, LFA-1/
ICAM-1, CD27:CD70, and CD2:CD48. CD40L is a member of the
TNF family and is transiently expressed on activated CD4⫹ T cells
(7, 8). The counter-receptor, CD40, is a member of the TNFR
family and is found on APCs, including B cells, bone marrowdendritic cells, follicular dendritic cells, and activated macrophages (7, 8). The CD40L:CD40 pathway was initially described
for its importance in B and T cell interactions, B cell activation, Ig
production, and isotype switching. Subsequently, CD40L:CD40
interactions were found to be essential for the initiation and activation of Ag-specific T cell effector functions and the activation of
costimulatory activity (including the up-regulation of B7 molecules) on APCs, including B cells, macrophages, and dendritic
cells (7). CD40L is expressed early in activation subsequent to the
engagement of the TCR with the MHC-peptide ligand complex on
the surface of the APC. CD40L transduces a signal to its counterreceptor, CD40, constitutively expressed on APCs, resulting in the
up-regulation of additional molecules involved in further T cell
costimulation (7, 9).
Administration of anti-CD40L mAb in vivo has been effective in
preventing collagen-induced arthritis and ameliorating GVHD in
murine models (10, 11). The in vivo administration of B cells from
CD40-deficient mice or the simultaneous administration of donor
splenocytes with anti-CD40L mAb has conferred alloantigen-specific tolerance to recipient mice (12, 13). In primates, administration of anti-CD40L mAb in vivo has been shown to prevent acute
renal and intrahepatic islet allograft rejection (14 –16). Although
the effect of in vivo administration of anti-CD40L mAb has been
impressive in preventing the rejection of solid organ allografts, we
0022-1767/00/$02.00
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A major goal of the transplant field is to selectively tolerize only those donor T cells recognizing host alloantigen and mediating
graft-vs-host disease (GVHD). Recently, we described an ex vivo approach in which the blockade of the CD40 ligand (CD40L):
CD40 costimulatory pathway in bulk MLR cultures induces donor CD4ⴙ T cells to become specifically tolerant to MHC class
II-disparate alloantigenic-bearing stimulators, resulting in a profound reduction in GVHD generation in vivo. In studies presented
in this work, we investigated the ex vivo requirements for tolerance induction. We found that CD4ⴙ T cells become profoundly
more hyporesponsive to alloantigen restimulation with prolonged culture duration such that 7 to 10 but not 4 days is needed to
achieve maximum alloantigen hyporesponsiveness as assessed in secondary MLR cultures and GVHD generation. By day 7, both
primed and tolerized cells had substantially increased blastogenesis and CD25 expression. Primed but not tolerized cells substantially down-regulated L-selectin expression, indicating that the tolerized cells do not become fully Ag experienced. Both Th1
and Th2 cytokine production is severely impaired by CD40L:CD40 blockade. Analysis of culture supernatants and results from
IL-4 and IL-10 knockout mice indicated that GVHD prevention was not mediated by a skewing toward a Th2 phenotype. The
addition of IL-4 to the cultures as a survival factor precluded the induction of tolerance in the anti-CD40L-cultured cells. These
data provide further impetus for the ex vivo use of anti-CD40L mAb to block GVHD generation. The Journal of Immunology,
2000, 164: 612– 622.
The Journal of Immunology
Materials and Methods
Mice
B6.C-H2bm12/KhEg (termed bm12), B10.D2 (H2d), and inbred C57BL/6
IL-10⫺/⫺ mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). C57BL/6 (termed B6) were purchased from the National Institutes of
Health (Bethesda, MD). (B6 ⫻ 129SV)F1 IL-4⫺/⫺ mice, backcrossed six
generations onto a B6 background, were obtained from Dr. Manfred Kopf
(Basel, Switzerland). bm12 and B6 (both H2b) mice differ at three amino
acids due to mutations in the IA region. Mice were used at 9 –10 wk of age.
All mice were housed in a specific pathogen-free facility in microisolator
cages.
In vitro MLR cultures
To purify B6 CD4⫹ T cells, axillary, mesenteric, and inguinal lymph nodes
were mashed, and single cell suspensions were passed through a wire mesh
and collected into PBS containing 2% FBS. Cell preparations were depleted of NK cells (hybridoma PK136, rat IgG2a, provided by Dr. Gloria
Koo, Rahway, NJ) and CD8⫹ T cells (hybridoma 2.43, rat IgG2b, provided
by Dr. David Sachs, Charlestown, MA) by coating with mAb, followed by
passage through a goat anti-mouse and goat anti-rat Ig-coated column (Biotex, Edmonton, Canada). The final composition of purified T cells was
determined by flow-cytometric analysis to be ⱖ94% CD4⫹ T cells. Responder B6 CD4⫹ T cells were mixed with irradiated (30 Gy), anti-Thy-1.2
mAb (hybridoma 30H-12, rat IgG2b, provided by Dr. David Sachs), and
anti-NK1.1 mAb plus baby rabbit complement (Nieffenegger, Woodland,
CA)-depleted bm12 splenic stimulators. Responder and stimulator cells
were suspended at a final concentration of 0.5 ⫻ 106/ml in 24-well plates
(Costar, Acton, MA) containing DMEM (BioWhittaker, Walkersville,
MD) with 10% FBS (HyClone, Logan, UT), 50 mM 2-ME (Sigma, St.
Louis, MO), 10 mM HEPES buffer, 1 mM sodium pyruvate (Life Technologies, Grand Island, NY), and amino acid supplements (1.5 mM Lglutamine, L-arginine, and L-asparagine) (Sigma) and antibiotics (penicillin, 100 U/ml; streptomycin, 100 mg/ml) (Sigma). Anti-CD40L mAb
(hybridoma MR1, hamster IgG) was obtained by culturing the hybridoma
in 10% FBS/DMEM in a hollow fiber bioreactor (AccucystJr, Cellex Biosciences, Minneapolis, MN). Supernatant was purified by ammonium sulfate precipitation. Anti-CD40L mAb was added at a final concentration of
50 ␮g/ml to primary MLR cultures. Plates were incubated at 37°C and 10%
CO2 for 4, 7, or 10 days. On day 5, the culture was fed 1:1 with new media
including mAb. To monitor primary MLR proliferation, 96-well roundbottom microtiter plates (Costar) were set up to contain 105 responders and
105 stimulators cells per well in the presence or absence of exogenous IL-2
(50 IU/ml) (Amgen, Thousand Oaks, CA). In some experiments, murine
IL-4 (Schering-Plough, Kenilworth, NJ) (sp. act., 1.5 ⫻ 107 U/mg) was
added at the indicated doses at the initiation of culture and readded at the
day 5 refeed.
To monitor secondary MLR proliferation, 3 ⫻ 104 washed, adjusted
responders and 105 irradiated (30 Gy) non-T cell-depleted stimulators were
plated in the presence or absence of IL-2 (50 IU/ml). Anti-CD40L mAb
was not present in the secondary MLR. Microtiter wells were pulsed with
tritiated thymidine (1 ␮Ci/well; Amersham Life Science, Buckinghamshire, U.K.) on the indicated days for 16 –18 h before harvesting and
counted in the absence of scintillation fluid on a ␤ plate reader (Packard
Instrument Company, Meriden, CT). Six wells were analyzed for each data
point.
Limiting dilution assay (LDA) cultures
Proliferating T lymphocyte precursor frequency analysis was accomplished
by setting up eight 3-fold serial dilutions of responder B6 CD4⫹ T cells at
30 replicates in 96-well round-bottom plates and incubating for 7 days with
irradiated (30 Gy), non-T cell-depleted bm12 splenic stimulators in the
absence of IL-2. Anti-CD40L mAb was added to the primary LDA at a
final concentration of 50 ␮g/ml. Secondary LDA were established with
washed, 10-day cultured control-primed or anti-CD40L-tolerized responder cells and bm12 or third-party B10.D2 splenic stimulators. AntiCD40L mAb was not present in the secondary LDA. Wells were pulsed
with tritiated thymidine for 18 h before harvesting and counted in the
absence of scintillation fluid on a ␤ plate reader. 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 calculated.
CTL determination
Washed 10-day cultured control-primed or anti-CD40L-tolerized cells
were incubated 4 h with 104 tritiated thymidine-labeled syngeneic or class
II-disparate Con A (Sigma)-stimulated splenocyte blasts at various E:T
ratios according to the JAM CTL assay (20). Wells, plated in triplicate,
were harvested and counted in the absence of scintillation fluid on a ␤ plate
reader. Percent lysis was calculated according to the equation: {[(cpm of
targets in absence of killers) ⫺ (cpm of targets in presence of killers)]/(cpm
of targets in absence of killers)} ⫻ 100.
GVHD induction
bm12 recipients were sublethally irradiated by exposing mice to 6 Gy total
body irradiation from a 137Cesium source at a dose rate of 85 cGy/min. Day
4, 7, or 10 MLR-cultured cells were injected i.v. at the doses indicated.
Peripheral blood was obtained by retroorbital venipuncture for measurement of day 14 and 28 hematocrit (HCT) values as an indicator of the bone
marrow-destructive effects of infused T cells.
Flow cytometry
Freshly purified and day 4, 7, and 10 MLR-cultured cells were assessed for
evidence of activation by forward scatter (FSC) and side scatter (SSC)
profiles and the coexpression of CD4 and activation Ags, including CD25,
L-selectin (CD62L), and CD40L. All studies were performed with twocolor flow cytometry using fluorescein- and PE-conjugated mAb (PharMingen, San Diego, CA). All results were obtained using a FACSCalibur
(Becton Dickinson, San Jose, CA). FSC and SSC settings were gated to
exclude debris. A total of 10,000 cells were analyzed for each
determination.
Quantitation of cytokine levels by ELISA
Murine cytokine levels in the supernatant 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.
Statistics
Survival data were analyzed by life-table methods, and actuarial survival
rates are shown. Group comparisons were made by log-rank test statistics.
For other data, group comparisons were made by Student’s t test. Values of
p ⱕ 0.05 were considered significant.
Results
Anti-CD40L mAb-cultured CD4⫹ T cells become profoundly
more hyporesponsive to alloantigen restimulation in vitro with
prolonged culture duration
Studies by other investigators have indicated that the induction of
anergy in mouse T cell clones, human T cell clones, and primary
T cells requires from 16 h up to 10 days of primary culture (5,
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found that the ex vivo tolerization of CD4⫹ T cells led to a more
profound reduction in GVHD lethality than did the in vivo administration of anti-CD40L mAb in the same setting (11, 17). In vivo
tolerance may be more difficult to achieve after BMT due to the 1)
induction of proinflammatory cytokines and increased expression
of costimulatory molecules on APCs as a result of the intense
conditioning protocols used for BMT (18, 19), and 2) difficulty of
achieving complete in vivo blockade at all possible sites of allorecognition. As an undesired side effect, the in vivo blockade of
costimulation could result in the induction of tolerance to tumor
Ags as well as alloantigens, preventing an effective graft vs tumor
effect.
We have previously described an ex vivo approach in which the
blockade of CD40L:CD40 interactions during a 10-day culture induces donor CD4⫹ T cells to become tolerant to host alloantigens,
resulting in a ⱖ30-fold reduction in GVHD lethality with no additional in vivo immunosuppression (17). Additionally, the tolerized bulk T cell population retained intact responses to Ags not
present during tolerization. Tolerance was long-lived and not
readily reversible in vivo. In these studies, we sought to determine
the prerequisites for tolerance induction in this ex vivo culture
system.
613
614
KINETICS OF ANTI-CD40L mAb EX VIVO TOLERIZATION
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FIGURE 1. Anti-CD40L mAb induces alloantigen hyporesponsiveness
in primary MLR culture, which is restored by exogenous IL-2. Primary
MLR culture consisted of B6 CD4⫹ lymph node T cell responders and
bm12 splenic stimulators plated in the absence (A) and presence (B) of
exogenous IL-2 (50 U/ml). Anti-CD40L mAb was added at 50 ␮g/ml at the
initiation of culture. On the y-axis are mean cpm ⫾ 1 SD of the mean. On
the x-axis are days in primary culture. A representative experiment is
shown.
21–26). We have previously reported a 10-day ex vivo tolerization
approach of primary murine CD4⫹ T cells, which results in profound secondary in vitro hyporesponsiveness and a ⱖ30-fold protection from GVHD mortality (17). In the studies presented in this
work, we sought to determine the minimum culture duration required for the induction of alloantigen hyporesponsiveness in vitro
and inhibition of GVHD in vivo. Purified B6 CD4⫹ lymph node T
cells were mixed with irradiated, T cell-depleted, MHC class IIdisparate bm12 splenic stimulators in the presence or absence of
anti-CD40L mAb. On days 4, 7, and 10, aliquots of cells were
washed to remove Ab and cytokines, and cells were either infused
in vivo into irradiated bm12 recipients or reexposed in vitro to
irradiated bm12 splenic stimulators in the absence of anti-CD40L
mAb. Fig. 1A illustrates the proliferative responses in the primary
culture. The addition of anti-CD40L mAb to the cultures progressively reduced proliferation by 21% (day 2), 91% (day 4), and 99%
(day 6) as compared with the control cultures. The addition of
exogenous IL-2 (50 U/ml) to the cultures prevented inhibition of
proliferation by anti-CD40L mAb, with both groups having equivalent proliferative responses (Fig. 1B). Fig. 2A–C illustrates the
secondary proliferative responses of the cultures established after
4, 7, or 10 days in primary culture in the presence of anti-CD40L
mAb. All secondary cultures were established in the absence of
anti-CD40L mAb and were derived from the same primary culture.
FIGURE 2. Anti-CD40L mAb-cultured CD4⫹ T cells become profoundly more hyporesponsive to alloantigen restimulation with prolonged
primary MLR culture duration. Secondary MLRs were established in the
absence of mAb after a 4 (A)-, 7 (B)-, or 10 (C)-day primary MLR culture
in the presence of anti-CD40L mAb. On the y-axis are mean cpm ⫾ 1 SD
of the mean. On the x-axis are days in secondary culture. A representative
experiment is shown.
Because our unpublished data have indicated that a 1- to 3-day
primary culture has an impaired capacity to mediate lethal GVHD
in vivo (P. A. Taylor and B. R. Blazar, unpublished observation),
we chose 4 days as the shortest culture period to study. Upon
reexposure to alloantigen restimulation in the secondary MLR, the
day 4 control culture peaked on day 3 instead of day 6 as in the
primary culture indicative of priming (Fig. 2A). Responses of cells
exposed to anti-CD40L mAb in the primary culture for 4 days
were inhibited by 45–74% during the first 3 days of the secondary
The Journal of Immunology
FIGURE 3. Anti-CD40L-tolerized B6 CD4⫹ T cells have reduced CTL
killing of bm12 targets. Ten-day control-primed or anti-CD40L-tolerized
B6 CD4⫹ T cells were washed and plated in a 4-h CTL assay with bm12
targets at various E:T ratios. On the y-axis is percent specific lysis ⫾ 1 SD.
On the x-axis is the E:T ratio.
regulated in only 10% of cells in the control cultures on day 4. By
day 7, both primed and tolerized cells had substantially increased
FSC, SSC (although values were higher in the primed cells as
compared with the tolerized cells), and CD25 expression (more
than 50% positive in both groups), indicating a greater degree of
activation than day 4 MLR cells (Fig. 4). Primed cells had substantially down-regulated or lost expression of L-selectin by day 7.
CD25 expression remained high on day 10 in both groups, with
62% positive in the anti-CD40L-cultured cells compared with 32%
positive in the control-primed cells. Notably, L-selectin remained
high in 85% of the tolerized cells, while more than one-half of the
primed cells lost expression (Fig. 4). We conclude from these data
that although both control- and anti-CD40L-cultured cells exhibit
marked evidence of blastogenesis and activation by day 7, the
tolerized cells do not become fully Ag experienced based upon
high L-selectin expression.
A 7- or 10- day but not 4-day blockade of the CD40L:CD40
interaction confers a high level of GVHD protection in vivo
To determine the length of time in culture required to induce tolerance induction in vivo, aliquots on days 4, 7, and 10 of the same
primary cultures used to establish the above secondary MLRs were
washed and infused into sublethally irradiated recipients bearing
the same alloantigen (bm12) used as stimulator cells in the primary
MLR culture. Data from our laboratory suggest cells cultured for
shorter periods (1– 4 days) have a reduced capacity to mediate
GVHD lethality (P. A. Taylor and B. R. Blazar, unpublished observation). Therefore, we infused 106, a higher number of cells, or
105 cells, which is typically lethal to all recipients. In contrast to
our previous data demonstrating no GVHD lethality with the infusion of 106 10-day anti-CD40L mAb-tolerized cells (17), a 4-day
culture period was insufficient to confer uniform GVHD protection. All recipients of 106 control-primed or anti-CD40L-cultured
cells died of GVHD-induced BM aplasia by day 25 after infusion
of cells (Fig. 5A). Sixty percent of mice receiving 105 anti-CD40Lcultured cells vs 20% of mice receiving 105 control-primed cells
survived the 65-day observation period ( p ⫽ 0.078 vs control). As
an indicator of donor CD4⫹ T cell-mediated GVHD-induced bone
marrow aplasia, HCT values were assessed in all mice on day 14
posttransfer of cells (Table I). There were no statistically signifi-
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culture. In secondary MLRs established after 7 days of primary
culture, the control group had an early vigorous response upon
reexposure to alloantigen, peaking on day 1 and gradually declining (Fig. 2B). This rapid secondary response by day 7 controlprimed cells has been reproducibly seen in these cultures. Responses of cells exposed to anti-CD40L mAb in the primary
culture for 7 days were inhibited by 71– 81% during the first 3 days
of the secondary culture. Seven-day anti-CD40L cultures were
more hyporesponsive upon alloantigen restimulation than 4-day
anti-CD40L cultures. The peak magnitude of the 7-day antiCD40L-cultured cells was 5400 cpm compared with 10,000 cpm
for the 4-day anti-CD40L-cultured cells. As compared with cultures established from 4 or 7 days of primary culture, secondary
MLR cultures established from 10-day anti-CD40L-cultured cells
were more profoundly hyporesponsive to alloantigen restimulation. At the time of peak secondary response, the proliferative
response of the 10-day anti-CD40L-cultured cells was inhibited by
95% as compared with the control-primed cells (Fig. 2C). The
peak magnitude of the 10-day anti-CD40L-cultured cells was 2700
cpm. These data indicate that hyporesponsiveness upon alloantigen
restimulation becomes more profound with prolonged ex vivo
blockade of the CD40:CD40L pathway. As in the primary MLR,
the addition of IL-2 fully restored secondary responses to alloantigen restimulation (data not shown).
As other measures of in vitro function, we determined the frequency of B6 CD4⫹ cells proliferating in response to bm12 alloantigen and the CTL killing of bm12 targets after 10 days of
primary culture with anti-CD40L mAb. Analysis of a 7-day LDA
established on day 0 of primary culture in the absence of IL-2
showed that the frequency of B6 CD4⫹ cells responding to bm12
alloantigen was 1:5,887 in the absence of mAb and 1:22,352 in the
presence of mAb, a 4-fold reduction. When LDAs were established with washed, 10-day cultured cells, the frequency increased
about 15-fold in the control group to 1:404. In contrast, the frequency in the anti-CD40L-tolerized cells remained the same as in
the primary culture (1:24,602). Although the frequency of antiCD40L mAb-tolerized B6 CD4⫹ responding to bm12 alloantigen
restimulation was decreased about 60-fold compared with the control-primed cells, the frequency of third party B10.D2 allo-responses was only decreased by less than 2-fold, indicating a high
degree of specificity (data not shown).
Because other investigators have reported a state of split anergy
in which anergic murine T cell clones can be rendered hyporesponsive to Ag reexposure in terms of proliferation and cytokine
production but have intact CTL (27, 28), we placed washed 10-day
control-primed cells and anti-CD40L-tolerized cells in a 4-h CTL
assay against bm12 Con A splenocyte blasts at various E:T ratios.
The percent specific lysis of bm12 targets by the 10-day antiCD40L-tolerized cells was approximately one-fourth that of the
control-primed cells (Fig. 3). Consistent with these data, there was
an 8 –10-fold reduction in the percentage of anti-CD40L-tolerized
cells staining positive for granzymes A and B mRNA (data not
shown). Granzymes, serine proteases stored in secretory CTL
granules, induce DNA fragmentation and CTL lysis of target cells
(29, 30). Therefore, a prolonged culture with anti-CD40L mAb
prevented the generation of optimal CTL effector function.
To determine whether anti-CD40L mAb treatment prevented T
cell activation, control- and anti-CD40L-cultured cells were evaluated by flow cytometry for their degree of blastogenesis and activation (Fig. 4). Although there was up-regulation of CD40L in
the control cultures by day 4, there was very little increase in cell
size (FSC) or internal complexity (SSC) at this time. Additionally,
CD25 expression was low in both groups. L-selectin (CD62L), a
homing receptor lost during activation (31), was marginally down-
615
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KINETICS OF ANTI-CD40L mAb EX VIVO TOLERIZATION
cant differences in HCT values between groups at either cell dose
from 4-day cultured cells. Collectively, these data indicate that 4
days of primary culture with anti-CD40L mAb are insufficient to
confer a high level of tolerance in vitro. Additionally, the relatively
moderate degree of hyporesponsiveness seen in secondary MLR
established from 4-day anti-CD40L-cultured cells did not translate
to protection from GVHD lethality in vivo.
In contrast, a 7-day MLR culture in the presence of anti-CD40L
mAb was sufficient to confer GVHD protection at the cell doses
used in these studies. All recipients of both cell doses of controlcultured cells (105 or 106) died of GVHD-induced BM aplasia by
day 23 (Fig. 5B). With the exception of one unusually late death at
the higher cell dose, all recipients of anti-CD40L-tolerized cells
survived the observation period (Fig. 5B) and had normal HCT
values at the time of elective sacrifice on day 65 (data not shown).
As expected, all recipients of 10-day anti-CD40L-tolerized cells
survived long term (Fig. 5C) and had normal HCT values at the
time of elective sacrifice (data not shown), while all recipients of
control-cultured cells died by day 16 (Fig. 5C). Although there
were no significant differences in GVHD mortality at the cell doses
used in this study between a 7- and a 10-day primary culture with
anti-CD40L mAb, the time to death and day 14 HCT values suggest that 10-day control-primed cells may have a greater GVHD
capacity than those cultured for 7 days. Mice receiving 105 10-day
control cells succumbed to GVHD mortality 1 wk earlier than
those receiving 105 7-day control cells ( p ⫽ 0.050). Additionally,
the day 14 HCT values in mice receiving 105 control cells suggest
a greater degree of aplasia with increasing culture duration (4-day
MLR HCT, 30.9; 7-day MLR HCT, 23.5; 10-day MLR HCT,
15.3). These data suggest that although a 7-day ex vivo tolerization
period confers protection from GVHD lethality, a 10-day ex vivo
tolerization period may confer even greater protection.
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FIGURE 4. Seven- and ten-day MLR cultures exhibit blastogenesis and activation. Primary MLR cultured cells were analyzed by flow cytometry on
days 0, 4, 7, and 10 for evidence of T cell activation. FSC and SSC overlay histograms are shown in the first two rows. The thin solid line represents the
control-cultured cells, and the bold line represents the anti-CD40L-cultured cells. The mean values are shown in the upper right of the histogram, with the
value of the control-cultured cells followed by that of the anti-CD40L-cultured cells. The last three rows illustrate overlay histograms of CD25, L-selectin,
and CD40L expression. The dotted line represents the negative isotype control; the thin solid line, the control-cultured cells; and the bold line, the
anti-CD40L-cultured cells.
The Journal of Immunology
617
Table I. Sublethally irradiated bm12 recipients of 7- or 10-day antiCD40L tolerized cells are protected from GVHD-induced BM aplasiaa
Culture Day
Group
No. of Cells Infused
HCT (%)
4
4
4
4
Control
Anti-CD40L
Control
Anti-CD40L
10
106
105
105
24.4 ⫾ 4.3
26.6 ⫾ 4.5
30.9 ⫾ 9.9
35.9 ⫾ 7.8
7
7
7
7
Control
Anti-CD40L
Control
Anti-CD40L
3 ⫻ 105
3 ⫻ 105
105
105
17.1 ⫾ 4.5
27.8 ⫾ 3.0*
23.5 ⫾ 7.2
27.8 ⫾ 2.0
10
10
10
10
Control
Anti-CD40L
Control
Anti-CD40L
3 ⫻ 105
3 ⫻ 105
105
105
14.0 ⫾ 2.8
28.1 ⫾ 5.9*
15.3 ⫾ 3.7
27.9 ⫾ 2.4*
6
a
Sublethally irradiated bm12 recipients (n ⫽ 5/group) were infused with the indicated number of cells from 4-, 7-, or 10-day control or anti-CD40L-treated cultures.
On day 14 postinfusion, hematocrit (HCT) values were determined in all surviving
mice. Average percent HCT is shown as mean ⫾ 1 SD.
*, p ⬍ 0.05, control vs. anti-CD40L cultures.
FIGURE 5. A 7-, but not 4-day primary MLR culture with anti-CD40L
mAb is sufficient to confer protection from GVHD lethality. Four-, 7-, or
10-day MLR cells cultured in the presence of anti-CD40L mAb were
washed and infused into sublethally irradiated bm12 recipients at the indicated cell doses (n ⫽ 5/group). On the x-axis are days posttransfer of T
cells. On the y-axis is the proportion of recipients surviving posttransfer.
(Four-day MLR, 106 cells, p ⫽ 0.209; 105 cells, p ⫽ 0.078; 7-day MLR,
3 ⫻ 105 cells, p ⫽ 0.0037; 105 cells, p ⫽ 0.0018; 10-day MLR, 3 ⫻ 105
cells, p ⫽ 0.0019; 105 cells, p ⫽ 0.0016.)
Anti-CD40L-cultured CD4⫹ T cells are not skewed toward a
Th2 phenotype
Fowler et al. (32) and Krenger et al. (40) have found that in vitro
generated, alloreactive Th2 (IL-4, IL-5, and IL-10) populations
have reduced GVHD capacity. To determine whether the GVHD
Table II. ELISA analysis of cytokine levels in supernatants from
primary MLR culturea
Th1
Primary
(day)
Group
IL-2
2
2
Control
Anti-CD40L
48
14
4
4
Control
Anti-CD40L
7
7
10
10
Th2
IFN-␥
IL-4
IL-10
0
0
0
0
0
0
286
27
23
0
0
0
0
0
Control
Anti-CD40L
312
17
187
9
0
0
0
0
Control
Anti-CD40L
111
9
281
6
0
0
0
0
a
Primary MLR cultures were established with B6 CD4⫹ T cells as responders and
irradiated, T cell-depleted bm12 splenic stimulators. Supernatants were harvested on
day 2, 4, 7, and 10 of primary culture. Final concentration of responders was 0.5 ⫻
106 cells/ml. Values are in pg/ml, and sensitivity is 1–10 pg/ml.
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protection of the anti-CD40L-cultured cells was mediated by a
skewing toward CD4⫹ Th2 cells, supernatants were taken from the
primary (days 2, 4, 7, and 10) and secondary cultures (days 1, 3,
and 5) and evaluated for the presence of Th1 (IL-2, IFN-␥) and
Th2 cytokines. Both control-cultured and anti-CD40L-cultured
cells produced IL-2 by day 2 of the primary MLR (Table II). IL-2
continued to accumulate in the control culture, peaking at 312
pg/ml on day 7. Cultures containing anti-CD40L mAb produced
12-fold less IL-2 (27 pg/ml vs 312 pg/ml) during the primary culture than the control. Peak amounts of IFN-␥ were detected at the
end of the primary culture in the control cells, with very low accumulation in the anti-CD40L cultures. The Th2 cytokines, IL-4
and IL-10, were not detected in the supernatants of either culture
in the primary MLR.
Secondary cultures established from both groups of 4-day primary cultures produced large amounts of IL-2 and IFN-␥ as early
as day 1 of culture (Table III). On a per cell basis, the cells exposed
to anti-CD40L mAb in the primary culture produced 10-fold more
IL-2 (915 pg/0.15 ⫻ 106 cells vs 312 pg/0.5 ⫻ 106 cells) and
6-fold more IFN-␥ (523 pg/0.15 ⫻ 106 cells vs 281 pg/0.5 ⫻ 106
cells) in the secondary MLR than the control group produced in the
primary MLR, suggesting significant Ag priming. In contrast to the
primary MLR, the Th2 cytokines, IL-4 and IL-10, were produced
618
KINETICS OF ANTI-CD40L mAb EX VIVO TOLERIZATION
Table III. ELISA analysis of cytokine levels in supernatants from
secondary MLR culturea
Th1
Primary
(day)
Th2
IL-2
IFN-␥
IL-4
IL-10
4
4
4
4
4
4
Control
Anti-CD40L
Control
Anti-CD40L
Control
Anti-CD40L
1
1
3
3
5
5
1171
560
1238
915
295
371
390
71
2592
414
2465
523
25
5
86
23
38
17
3
0
237
57
641
108
7
7
7
7
7
7
Control
Anti-CD40L
Control
Anti-CD40L
Control
Anti-CD40L
1
1
3
3
5
5
1193
286
279
53
154
16
2729
10
3310
48
3268
25
103
4
59
7
30
5
98
7
1586
35
1061
47
10
10
10
10
10
10
Control
Anti-CD40L
Control
Anti-CD40L
Control
Anti-CD40L
1
1
3
3
5
5
1167
33
698
6
133
4
3253
4
3368
2
3190
3
87
22
125
1
46
0
53
0
879
4
1308
16
a
Secondary MLR cultures were established in the absence of anti-CD40L mAb
with 4-, 7-, or 10-day cultured B6 CD4⫹ T cells as responders and irradiated, non-T
cell-depleted bm12 splenic stimulators. Supernatants were harvested day 1, 3, and 5
of secondary culture. Final concentration of responders was 0.15 ⫻ 106 cells/ml.
Values are in pg/ml and sensitivity is 1–10 pg/ml.
by the 4-day control-primed cells upon in vitro alloantigen restimulation, indicating that the control bulk culture consisted of a
mixed population of Th1- and Th2-primed cells (Table III). Although in secondary MLRs, Th2 cytokines by the anti-CD40Lcultured cells were reduced in comparison with the control-primed
cells, there was still substantial production. These data indicated
that although a 4- day culture in the presence of anti-CD40L mAb
reduced cytokine production upon reexposure to alloantigen in
comparison with the control cultures, there was still a vigorous
Th1 and Th2 cytokine response to alloantigen restimulation. Given
the high levels of IL-2 production by the 4-day antiCD40L-cultured cells, it is perhaps not surprising that they did
mediate GVHD lethality in vivo (Fig. 5A).
IL-2 and IFN-␥ production in secondary cultures established
from 7-day anti-CD40L cultures was reduced 3-fold (286 pg/ml vs
915 pg/ml) and 11-fold (48 pg/ml vs 523 pg/ml), respectively, as
compared with that established from 4-day anti-CD40L cultures. It
is interesting to note that although the IL-2 production was reduced
75% as compared with the 7-day control group, there was more
IL-2 produced on a per cell basis by the 7-day anti-CD40L cultures
in secondary MLR than in the primary MLR control group. Surprisingly, in spite of this substantial IL-2 production in vitro upon
Ag restimulation by the anti-CD40L-cultured cells, these cells did
not mediate GVHD in vivo (Fig. 5B). IFN-␥ production by the
7-day anti-CD40L cultures was reduced by 99% compared with
the control-primed cultures. As with the Th1 cytokines, there were
lower levels of Th2 cytokines produced in secondary MLRs by
7-day anti-CD40L-cultured cells than by 4-day cultures. Th1 and
Th2 cytokine production in secondary cultures established from
10-day anti-CD40L cultures were the most profoundly suppressed
(Table III). Most notably, IL-2 production in secondary MLR established from 10-day anti-CD40L cultures was 9-fold (33 pg/ml
vs 286 pg/ml) lower than that from 7-day anti-CD40L cultures.
Overall, we found a pronounced and progressive dimunition of
Th1 and Th2 cytokine production in the secondary MLRs of the
anti-CD40L cultures with increasing primary culture duration.
FIGURE 6. B6 IL-4 knockout CD4⫹ T cells are susceptible to tolerance
induction via a 10-day ex vivo tolerization culture with anti-CD40L mAb.
A, Primary MLR culture consisted of B6 IL-4 knockout CD4⫹ T cells and
bm12 splenic stimulators. Anti-CD40L was added at 50 ␮g/ml at the initiation of culture. On the y-axis are mean cpm ⫾ 1 SD of the mean. On the
x-axis are days in primary culture. B, On day 10 of primary culture, the
above MLR was washed and infused into sublethally irradiated bm12 recipients at 105 cells/mouse (n ⫽ 8/group). On the x-axis are days posttransfer of T cells. On the y-axis is the proportion of recipients surviving
posttransfer.
Because ELISA measures the total amount of immunologically
reactive protein present in the supernatants, but doesn’t take into
account cytokine consumption, Th2 skewing may have been
present, but not at a level detectable by ELISA. As additional
evidence that GVHD protection was not due to a skewing toward
a Th2 phenotype, we used CD4⫹ cells from IL-4 or IL-10 knockout mice as responders in our 10-day ex vivo tolerization MLR
cultures. Ex vivo blockade of the CD40L:CD40 pathway via antiCD40L mAb in IL-4 knockout CD4⫹ cells resulted in decreased
proliferation in the primary MLR (Fig. 6A) and profound hyporesponsiveness upon alloantigen restimulation in vitro (data not
shown). Mice receiving 105 control-primed IL-4 knockout CD4⫹
cells died of BM aplasia by 3 wk. Mice receiving 105 anti-CD40Lcultured IL-4 knockout CD4⫹ cells survived the 65-day observation period (Fig. 6B) and had normal HCT at time of elective
sacrifice (data not shown). Similar results were found with IL-10
knockout CD4⫹ cells. Proliferation in primary and secondary
MLRs was profoundly reduced in anti-CD40L-cultured IL-10
knockout CD4⫹ cells (data not shown). Mice receiving 105 antiCD40-cultured IL-10 knockout CD4⫹ cells survived the observation period (Fig. 7). In contrast, mice receiving 105 control-primed
IL-10 knockout CD4⫹ cells died of BM aplasia by 2 wk posttransfer. These data indicate that production of the Th2 cytokines, IL-4
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Group
Secondary
(day)
The Journal of Immunology
619
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FIGURE 7. B6 IL-10 knockout CD4⫹ T cells are susceptible to tolerance induction via a 10-day ex vivo tolerization culture with anti-CD40L
mAb. Ten-day MLR cultures were washed and infused into sublethally
irradiated bm12 recipients at 105 cells/mouse (n ⫽ 8/group). On the x-axis
are days posttransfer of T cells. On the y-axis is the proportion of recipients
surviving posttransfer.
or IL-10, was not required for anergy induction or GVHD
protection.
The addition of IL-4 to the cultures precluded the induction of
anergy in the anti-CD40L-cultured cells
IL-4 has been reported to preclude anergy induction by signaling
through the common ␥-chain of the IL-2R, IL-4R, IL-7R, and IL15R (21, 33, 34). IL-4 has also been found to prevent cell death
without cell division and proliferation by preventing the decay of
bcl-2 in activated T cells (35). Because cell recovery in the 10-day
anti-CD40L cultures is relatively low (ⱕ10% of starting cell number), we examined the effect of a day 0 addition of a low concentration IL-4 as a survival factor to determine whether we could
improve recovery without precluding the induction of anergy. The
addition of 0.1 ng/ml of IL-4 did not reproducibly improve recovery. However, the addition of 1 ng/ml or 10 ng/ml of IL-4 added
to the cultures on day 0 did reproducibly and favorably increase
recovery from ⬍10% to 32–39% of the starting cell number (data
not shown). The addition of 1 ng/ml of IL-4 added to cultures on
day 0 did not preclude the inhibition of proliferation by antiCD40L mAb in the primary MLR (Fig. 8A). Peak proliferation in
the primary culture was inhibited by anti-CD40L mAb by 96%
even in the presence of 1 ng/ml IL-4. Cell recovery on day 10 was
8% in the anti-CD40L cultures in the absence of IL-4 and 48% in
the presence of IL-4. Despite a high degree of blockade of proliferation in the primary MLR cultures containing both anti-CD40L
mAb and IL-4, these cells were only modestly hyporesponsive
upon in vitro alloantigen restimulation in the secondary MLR (Fig.
8B). This modest reduction (⬍40%) in secondary response as compared with the control-primed cells did not translate to GVHD
protection in vivo. All recipients of either 105 control-primed/IL-4
or anti-CD40L/IL-4-cultured cells died of GVHD-induced BM
aplasia by day 18 after cell transfer (Fig. 8C). In contrast, all recipients of 105 anti-CD40L-cultured cells in the absence of supplemental IL-4 survived the 2-mo observation period. These data
indicate that IL-4 (1 ng/ml) added as a survival factor on day 0
precluded the induction of anergy.
Discussion
In this study, we have demonstrated that anergy induction via in
vitro blockade of the CD40:CD40L costimulatory pathway be-
FIGURE 8. Although the addition of IL-4 at 1 ng/ml at the initiation of
culture did not interfere with the inhibition of primary MLR by antiCD40L mAb, the cells were only modestly hyporesponsive upon alloantigen restimulation in the secondary MLR and were not tolerant in vivo. A,
Primary MLR culture consisted of B6 CD4⫹ lymph node T cell responders
and bm12 splenic stimulators in the presence of IL-4 (1 ng/ml). AntiCD40L mAb was added at 50 ␮g/ml at the initiation of culture. B, Secondary MLRs were established from the above 10-day primary MLR cultures in the absence of IL-4 and anti-CD40L mAb. On the y-axis are mean
cpm ⫾ 1 SD of the mean. On the x-axis are days in secondary culture. C,
Ten-day MLR cultures established in the presence or absence of IL-4 were
washed and infused into sublethally irradiated bm12 recipients at 105 cells/
mouse (n ⫽ 5/group). On the x-axis are days posttransfer of T cells. On the
y-axis is the proportion of recipients surviving posttransfer.
came more profound with increasing duration in primary culture as
measured by in vitro secondary MLR proliferative responses, cytokine production, and in vivo GVHD protection. Th1 and Th2
KINETICS OF ANTI-CD40L mAb EX VIVO TOLERIZATION
cytokine production were both reduced in the primary and secondary MLRs of the anti-CD40L-cultured cells, indicating that GVHD
was not mediated by a skewing toward a Th2 phenotype. Additionally, IL-4 and IL-10 knockout CD4⫹ cells were susceptible to
the induction of alloantigen hyporesponsiveness in our model, further indicating that Th2 cytokines were not obligatory. IL-4 added
to the cultures at doses that improved cell recovery precluded the
induction of anergy, indicating that IL-4 does not mediate but
rather can prevent the induction of tolerance.
We have previously demonstrated a profound reduction in
GVHD lethality with a 10-day ex vivo tolerization culture duration
(17). Because studies by others have indicated that the time to
optimal tolerance induction can vary between 16 h and 10 days, we
wished to determine whether this relatively prolonged duration
was essential for GVHD protection (5, 21–26). Our data indicated
a 4-day MLR culture in the presence of anti-CD40L mAb was
insufficient to induce tolerance. Although CD40L expression was
up-regulated by day 4 of control cultures, cells had not substantially increased in size and internal complexity, CD25 expression
was not increased, and L-selectin had not yet significantly downregulated. Together these data may suggest that T cells require a
certain level of activation for tolerance induction to occur.
In contrast to the partial inhibitory effect of 4-day anti-CD40L
MLR cultures, 7 days of primary culture with mAb were sufficient
to induce hyporesponsiveness in vitro and tolerance in vivo, while
10 days of culture resulted in a more profoundly tolerized population of cells. Although the cell doses used for in vivo GVHD
generation did not allow us to conclude that 10 days of primary
culture led to greater in vivo GVHD prevention than a 7-day primary culture, the secondary MLR and cytokine data indicated that
the secondary in vitro hyporesponsiveness was more profound
with a 10-day primary tolerization culture duration. In all experiments, there was a small burst of proliferation on day 1 of secondary MLR by these cultures. This slight degree of proliferation
in the hyporesponsive cells in the secondary MLR was accompanied by substantial IL-2 production especially from secondary cultures established from 7-day tolerized MLR cultures. Nine-fold
more IL-2 was produced in response to alloantigen restimulation
by 7-day tolerized cultures as compared with 10-day tolerized cultures. Based upon the high levels of IL-2 detected in the supernatants of secondary cultures of tolerized cells established from
7-day MLR cultures, one might have predicted greater GVHD
mortality. However, this early proliferation and IL-2 production by
the tolerized population were not sustained and rapidly declined.
Although we do not yet know whether the Ag-specific cells in the
tolerized cultures were deleted, we hypothesize that deletion is
probably not the sole mechanism involved, as the bulk population
had a partial early proliferation and produced IL-2 in response to
alloantigen restimulation.
The degree of hyporesponsiveness seen in the secondary MLR
upon reexposure to alloantigen correlated well with GVHD protection in vivo, suggesting that the in vitro assays could potentially
be used as a monitoring device, which might be predictive of the
clinical outcome. The primary MLR may be less predictive of
successful tolerance induction than the secondary MLR (see Fig.
8A). Although the degree of blockade of proliferation was very
high by anti-CD40L mAb plus IL-4 in the primary MLR, there was
only modest secondary hyporesponsiveness and no GVHD
protection.
The requirement for a relatively long primary culture duration to
induce alloantigen hyporesponsiveness is in contrast to studies by
other investigators. Taub et al. found that a 3-day incubation of the
phosphatidylinositol-3-kinase inhibitor, wortmanin, was sufficient
to induce alloantigen-specific tolerance in a murine GVHD model
(22). However, as the degree of GVHD protection was not quantified, it is unknown whether a longer incubation would have resulted in a more profound resistance to GVHD induction. Although it is possible that tolerance induction via small intracellular
signaling inhibitors may occur faster than that via Ab blockade of
cell surface costimulatory molecules, not all surface-mediated tolerance-inducing events require prolonged ex vivo culture. For example, Jenkins and Schwartz found that only a 16-h incubation of
pigeon cytochrome c-specific T cell clones with Ag and chemically modified splenocytes was sufficient to induce unresponsiveness to restimulation with normal APC and Ag (5). Although these
studies used T cell clones and a peptide Ag and not bulk CD4⫹
lymph node and alloantigen as ours do, it is tempting to speculate
that the relatively short culture duration needed to tolerize these
cells may be due to the fact that APCs in their culture system were
completely incapable of up-regulating the cell surface expression
of all potential costimulatory molecules. In our system, costimulatory molecules other than those entirely dependent upon induction via CD40L:CD40 interaction potentially could be induced to
be expressed and therefore may have transduced some costimulatory signal. Alternatively, their model may involve a higher frequency of responding T cells and a more rapid response requiring
a shorter culture duration to induce anergy. Optimal anergy induction via blockade of costimulation may require a culture duration
of sufficient length to ensure that all potentially alloreactive cells
have had maximal opportunity for TCR engagement, initial activation and up-regulation, and subsequent blockade of CD40L. Although CD40L was up-regulated by day 2, peak proliferative responses occurred on day 5 or 6 of primary culture. Additionally,
maximal up-regulation of the activation Ag, CD25, was not seen
until day 7 of primary culture, with very low expression by day 4.
Blockade of costimulation as a strategy to induce anergy induction
may be required throughout the duration of these events. The minimum culture duration required to obtain optimal anergy may depend as much on the kinetics of the response as on the strategy
used to induce anergy.
In a model more analogous to ours, Gribben et al. found that
maximal hyporesponsiveness of human alloreactive T cells to alloantigen restimulation was achieved with 36 h via blockade of the
CD28:B7-1/B7-2 pathway (21). Other studies have indicated that
a longer culture duration is necessary or at least beneficial. Two
studies found that a minimum of 5- to 7-day culture of human T
cells with stimulator cells in the presence of anti-B7-1 and cyclosporine, followed by 1- to 2-day rest was needed to yield a hyporesponsive state (23, 26). In a system in which human alloreactive
T cells were rendered hyporesponsive by IL-10, T cells were noted
to become progressively more hyporesponsive during a 3- to 10day culture period (25). These data and ours suggest that hyporesponsiveness becomes increasingly profound with increasing culture duration in bulk cultures consisting of naive, potentially
alloreactive T cells.
We have previously demonstrated that OVA-specific T cells exposed to anti-CD40L mAb during tolerization to alloantigen have
intact OVA responses at optimal Ag concentrations (17). Consistent with these data, although the frequency of responding T cells
to relevant alloantigen was decreased by 60-fold in anti-CD40Lcultured cells as compared with the control-cultured cells, the frequency of the tolerized cells to third party alloantigen was decreased only 2-fold. This suggests that a bulk T cell population that
is tolerized ex vivo to alloantigen may still be capable of responding to viral or tumor Ags encountered in vivo.
In contrast to other groups reporting split anergy in which proliferation and cytokine production were diminished, but cytotoxicity was not impaired (27, 28), we found CTL killing by the
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620
The Journal of Immunology
Acknowledgments
We thank Dr. Arlene Sharpe for critical review of the manuscript.
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anti-CD40L-cultured cells was approximately one-fourth that of
the control-primed cells. Perhaps more importantly, anti-CD40Lcultured cells failed to induce mortality in mice, while mice receiving control-primed cells died of BM aplasia. Consistent with
this, IFN-␥ required for CTL generation was profoundly reduced
in both the primary and secondary MLR of the antiCD40L-cultured cells. We and others have found that an intact
CD40L:CD40 pathway is required for optimal in vivo CTL generation, and that the administration of anti-CD40L mAb abrogates
a GVL effect (36 –39) (P. A. Taylor and B. R. Blazar, unpublished
observation). Collectively, our data indicate that the anti-CD40Lcultured cells are hypofunctional in terms of proliferation, cytokine
production, and cytotoxicity.
Although Th2 cells have been reported in some (32, 40) but not
all (41) studies to result in reduced GVHD as compared with Th1
cells, our hyporesponsive cells were not skewed toward a Th2
phenotype. The anti-CD40L-cultured cells made far less of the Th2
cytokines, IL-4 and IL-10, upon alloantigen restimulation than did
the control-primed cells. As well as being a Th2 cytokine, IL-10 is
an immunosuppressive cytokine that has been shown to induce
tolerance in human alloreactive T cells (25). In another study, endogenously produced IL-10 and TGF-␤ were found to mediate
superantigen-induced tolerance in a murine in vivo model (42).
However, both IL-4 and IL-10 knockout CD4⫹ cells were susceptible to anergy induction in our model, indicating there is not an
absolute requirement for either of these cytokines for hyporesponsiveness in vitro and GVHD protection in vivo. Additionally, we
did not find free TGF-␤, another immunosuppressive cytokine, in
the supernatants of either culture group in either the primary or the
secondary MLR (data not shown). We did find the chemokines,
RANTES and macrophage-inflammatory protein 1-␣, present in
the supernatants of the anti-CD40L cultures in both the primary
and the secondary MLR, indicating that the tolerized cells are not
globally, metabolically disabled (data not shown).
Because cell recovery is typically low (ⱕ10%) in the anergic
cultures, we examined the effect of the addition of IL-4 as a survival factor to the primary cultures in an attempt to increase our
cell recovery. We did not uncover a concentration of IL-4 that both
increased recovery and permitted the induction of secondary hyporesponsiveness. As IL-7 also has been reported to be involved
with T cell survival in vivo and in vitro (35), we examined the
addition of IL-7 on cell recovery and induction of secondary hyporesponsiveness and discovered that a dose or schedule that improved cell recovery also precluded anergy induction (P. A. Taylor
and B. R. Blazar, unpublished data). We surmise that the addition
of IL-4 or IL-7 at concentrations high enough to increase recovery
results in sufficient signaling through the IL-2R common ␥-chain
to preclude the induction of anergy.
These studies indicate that ex vivo anti-CD40L tolerization becomes more profound with increasing primary culture duration,
highlighting the importance of kinetics studies in tolerance-induction protocols. Our data indicate that a combination of flow cytometry and in vitro secondary proliferative, cytotoxic, and cytokine responses may be useful indicators of the efficacy of a
tolerizing procedure in preventing GVHD generation in vivo. AntiCD40L mAb ex vivo tolerization warrants consideration as a potential therapeutic modality for the prevention of GVHD.
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