1. Art and Diseño

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Coblockade of the LFA1:ICAM and CD28/CTLA4:B7 Pathways
Is a Highly Effective Means of Preventing Acute Lethal Graft-Versus-Host
Disease Induced by Fully Major Histocompatibility
Complex-Disparate Donor Grafts
By Bruce R. Blazar, Patricia A. Taylor, Angela Panoskaltsis-Mortari, Gary S. Gray, and Daniel A. Vallera
We have developed an in vitro system in which C57BLl6
donor splenocytes are exposed t o B1O.BR host alloantigens
in the context of deficient CD28:B7 signaling as a means of
preventing graft-versus-host disease (GVHD).Although 54%
t o 82% of MLR alloresponse was inhibited by cytotoxic Tlymphocyte antigen 4 (CTLA4I-lg treatment of host stimulator cells, treated splenocytes were still capable of causing
GVHD when infused in vivo. By adding anti-leukocyte function antigen 1 (anti-LFAl) antibody t o hCTLA4-lg in vitro
t o coblock the LFA1:intercellular adhesion molecule (ICAM)
signaling, splenic alloresponse was inhibited by 28996, yet
GVHD induction capabilities were retained. Because antigen-primed cells might be more susceptible t o CD28:B7
blockade, we investigated whether hCTLA4-lg alone, antiLFAl antibody alone, or the combination of both added t o
donor-antihost in vitro primed cells could reduce GVHD. To
facilitate hyporesponsiveness induction and t o block B7 and
ICAM ligands that are upregulated during GVHD, these reagents were also administered t o recipients post-BMT. We
have shown thathCTLA4-lg plus anti-LFAl antibodyis highly
effective in preventing GVHD-inducedl e t h a l i i (88% t o 100%
of treated mice surviving versus 0% t o 28% of controls surviving). For optimal prevention, both hCTLAClg and antiLFAl must beused in vitro in the context of donor-antihost
primed splenocytes and continued in vivo. This in vitro-in
vivo combined approach was associated with donor engraftment, and recipients were not globally immunosuppressed. We conclude that blocking both theCD281B7 and
the LFA1:ICAM pathways are critical t o effective GVHD prevention and may offer advantages t o in vitro donor T-cell
removal.
0 1995 by The American Society of Hematology.
T
marrow transplant (BMT), the donor splenocytes would be
exposed to CTLACIg before these cells would have been
primed to host alloantigens. Studies by Turka et al16.’’would
suggest that primed cells are more susceptible to CTLA4-Ig
infusions than nonprimed cells. Many of the reports of anergy induction have involved the testing of antigen-specific
(ie, antigen-primed) T-cell clones. Whereas in vivo antigenspecific hyporesponsiveness has been described, a number
of these studies have used model systems that may not be
applicable to the transfer of allogeneic lymphohematopoietic
cells such as transgenic mice analyzed in a resting state’.’8
or murine recipients of xenografted islets.” In contrast with
these examples, GVHD induced in lethally irradiated recipients generates a fulminant systemic reaction characterized by
massive tissue destruction, release of inflammatory cytokines
and T-cell responses to alloantigens present on multiple tis-
CELLS ARE typically activated in vivo by the binding
of antigendpeptides to the T-cell receptor (TCR). For
an efficient and sustained T-cell response to occur, a costimulatory signal, usually provided by professional antigenpresenting cells (APCs), must be transduced to the T cell.’
Using antigen-specific Thl clones, Jenkins and Schwartz’
first showed that T cells exposed to antigerdpeptide in the
presence of paraformaldehyde-fixed APCs may become incapable of responding when reexposed to the antigen/peptide, a state known as anergy. This approach has been shown
to inhibit the ability of resting APCs to costimulate T cells
by preventing the upregulation of CD2WCTLA4 ligands that
occur during the immune response and by precluding efficient interleukin-2 (IL-2) production via the CD2WCTLA4
p a t h ~ a y .A~ .soluble
~
fusion protein, consisting of the extracellular domain of CTLA4 and an Ig moiety, which specifically blocks the CD28/CTLA4:B7 costimulatory pathway,
similarly renders T cells anergic to antigen/peptides present
during the blockade.””’ Because CD28/CTLA4:B7 blockade reduces the primary mixed lymphocyte reaction
(MLR),’’.’3strategies that inhibit this pathway could be used
to make tolerant the small subset of donor T cells with
host alloreactive capacity and be exploited as a means of
preventing lethal GVHD.
Our group has shown that in vivo blockade of one such
T-cell costimulatory pathway, CD28/CTLA4:B7, was effective in reducing, but not eliminating lethal GVHD in irradiated recipients of fully allogeneic or minor histocompatibility-only disparate donor grafts.I4.l5In those experiments,
mice were given a fusion protein consisting of the extracellular domain of human CTLA4 fused with a murine (m) or
human (h) Ig heavy chain (termed CTLA4-Ig) to extend
circulating half-life of the recombinant protein. There are
several reasons why incomplete anti-GVHD responses to
CTLA4-Ig infusions might have been observed in these studies. One possibility is that the timing of CTLA4-Ig infusions
wasnot optimal for tolerance induction in vivo. Because
hCTLA4-Ig infusions were initiated on days 0 or - 1 of bone
Blood, Vol 85, No 9 (May l ) , 1995: pp 2607-2618
From the Department of Pediatrics, Division of Bone Marrow
Transplantation, and the Department of Therapeutic Radiology, Section on Experimental Cancer Immunology, University of Minnesota
Hospital and Clinic, Minneapolis, MN; and RepliGen Corp, Cambridge, MA.
Submitted August 26, 1994; accepted December 13, 1994.
Supported in part by US Public Health Service Grants No. ROIA134495, ROI-CA 31618, and Pol-AI35296 awarded the
byNational
Cancer Institute and the National Institute of Allergy and Infectious
Diseases, Department of Health and Human Services. B.R.B. is a
recipient of the Edward Mallinckrodt Jr Foundation Scholar Award.
This is no. 33 in a series on murine bone marrow transplantation
across the mixed lymphocyte reaction.
Address reprint requests to Bruce R. Blazar, MD, Box 109, University of Minnesota Hospital and Clinic, 420SE Delaware St, Minneapolis, MN 55455.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I995 by The American Society of Hematology.
0006-4971/95/8509-05$3.00/0
2607
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BLAZAR ET AL
2608
sue targets.’” The graft-versus-host reaction may be less susceptible to anergy induction by CTLA4-Ig by virtue of the
systemic inflammatory processes that accompany irradiation
and alloreactivity.
Another possibility is that other APC:T-cell pathways capable of providing this costimulation might override the beneficial anti-GVHD effect of CTLA4-Ig infusions. Some of
the candidates include LFAl :intercellular adhesion molecule
(ICAM),’3.2’CD2:CLM8,” CD40 ligand:CD40,23.24
and the
VLA4:VCAMl,” and interactions. In vivo blockade of the
first two receptor:counter-receptor pairs has been shown to
facilitate the acceptance of solid organ grafts,”.” whereas
blockade of the last three receptor:ligand pairs23-26
has resulted in diminished antigenic responses, including GVHD,
in vivo.
The present study was undertaken for two reasons: (1) to
determine if donor cells exposed to host alloantigens in vitro
before BMT in the presence of a CD28/CTLA4:B7 blockade
could be made tolerant of host alloantigens in vivo resulting
in a diminished GVHD capacity and, if not, to determine
whether continuing the blockade strategy in vivo would be
advantageous; (2) to determine if the coblockade of an alternative T-cell signaling pathway, such as LFAl:ICAM, would
be additive or synergistic to the blockade of the CD28/
CTLA4:B7 pathway in preventing GVHD in lethally irradiated recipients of fully allogeneic donor grafts, and to determine whether long-term surviving recipients would become
severely immunosuppressed. Toward that end, we have examined mice during the graft-versus-host reaction in vivo
for upregulation of B7and ICAM ligands to decide if
LFA1:ICAM might be an appropriate determinant for
coblockade along with CD28:B7 and to decide whether in
vivo infusion of hCTLA4-Ig in conjunction with anti-LFAl
antibody would be a reasonable approach to augment an in
vitro procedure to reduce GVHD alloreactivity in vivo.
MATERIALS AND METHODS
Mice. B IO.BR/SgSnJ (H-2k)recipient mice were purchased from
Jackson Laboratory (Bar Harbor, ME). C57BLl6 (H-2b) donor mice
were purchased from the National Institutes of Health (Bethesda,
MD). Donors and recipients were female. Donors were 4 to 6 weeks
old and recipients were 8 to I O weeks old at the time of BMT. In
addition, C.H-2h”’2(designated bm12) congeneic mice, which differ
from C57BW6 by virtue of a 3-amino acid mutation in the MHC
class I1 locus, were obtained from Jackson Laboratory and used as
a source of stimulator cells in some in vitro assays.
BMT. Our transplant protocol has been described in detaiLZ7
BIO.BR recipients were conditioned with 8.0 Gy total body irradiation administered from a Philips RT 250 Orthovoltage Therapy Unit
(Philips Medical Systems, Brookfield, W)filtered through 0.35-mm
Cu at a final absorbed dose rate of 0.41 Gray (Gy)/min at 225 kV
and 17 mA. Donor BM was collected into RPMI 1640 medium by
flushing it from the shafts of femurs and tibias. Recipients (8 mice
per group per experiment) received 25 X lo6 BM cells from C57BLl
6 donors that had been T-cell depleted with anti-Thyl.2 monoclonal
antibody (MoAb) (hybridoma 30-H-12, rat IgG2b, provided by Dr
David Sachs, Massachusetts General Hospital, Cambridge) plus C‘
as previously
Single cell suspensions of splenocytes
were obtained (as a source of GVHD-causing effector cells) by
passing minced spleens through a wiremeshand collecting them
into RPMI 1640. Noncultured or in vitro cultured splenocytes (as
indicated) were suspended with anti-Thy1.2 plus C’-treated BM, and
0.5 mL volume containing 25 X 10‘ anti-Thy1.2 plus C‘-treated BM
plus 25 X IOh splenocytes was infused. This dose of splenocytes
was chosen to provide GVHD-induced mortality in at least 70% of
recipients (see Results). The final composition of T cells in this
donor graft ranged from 10%to 20% of theentire inocula, simulating
the lower spectrum of the content of T cells contained with human
BM aspirates used for human BMT. In this latter setting, peripheral
blood T cells typically contaminate donor BM aspirates.3” Asan
additional control, some mice received splenocytes that were depleted of T cells with anti-Thyl.2 plus C’ as described above.
hCTLA4-Ig protein and anti-LFAI MoAb preparation and usage.
hCTLA4-Ig waspurified from supernatants ofprotein A Chinese
hamster ovary cells electroporated with an expression vector containing the extracellular portionof hCTLA4 joined tothe CHIHinge-CH2-CH3 domains derived from a human genomic IgGl gene
with immobilized protein A.” Purified hCTLA4-Ig consisted entirely
of the dimeric form. Anti-LFAl (anti-CD1la) (hybridoma FD441 .S,
rat IgG2b.kindly provided by Dr Frank Fitch, University of Chicago,
Chicago IL) MoAb was used as purified (greater than 85% pure as
assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel) ascites fluid obtained after ammonium sulfate precipitation
and dialysis as previously described.” For in vivo administration,
mice were injected intraperitoneally with phosphate-buffered saline
(PBS), hCTLA4-Ig (250 pgldose on days - I and 0, and then 100
&dose thrice weekly until day 28 post-BMT according to our previous dose optimization studiesI4),and/or anti-LFAl MoAb (300 pgl
dose beginning on day - 1 and continuing twice weekly through day
29 post-BMT to ensure adequate in vivo blockade of LFA1:ICAM
binding).
In vitro incubation of splenocytes with hCTLA4-Ig a d o r antiLFAl MoAb. Donor C57BL/6 splenocytes and irradiated (30 Gy)
hostBIO.BR splenocytes were concentrated to 8 X 10‘ cells/mL
each in Dulbecco’s minimal essential medium (Bio Whitttaker,
Walkersville, MD), 10% fetal calf sera (Hyclone, Logan, UT), 2
mom) (Sigma Chemical CO, St Louis,
mercaptoethanol ( 5 X
MO), 10
mmoVL
HEPES buffer, 1 mmol/L sodium pyruvate
(GIBCO BRL, Grand Island, NY), and amino acid supplements
(1.5 mmol/L. L-glutamine, L-arginine, and L-asparagine) (Sigma),
antibiotics (penicillin: 1 0 0 UlrnL, streptomycin: 100 pg/mL)
(Sigma), and placed at 37°C and 10%CO2.For scale-up experiments,
in which the incubated cells were infused in vivo, donor (8 X 10‘
cellslml) and irradiated host splenocytes (8 X IO6 cellslml) were
added together and placed into 225-cm2 flasks (Costar, Cambridge,
MA), and incubated in 10% CO2 for 3 days. In some experiments,
an aliquot of cells was removed after the completion of the culture
period for assessment of proliferation and/or phenotyping by flow
cytometry as described in detail below.
For experiments with naive splenocytes, hCTLA4-Ig (SO pLg/mL)
was added on the day of culture initiation (day 0) before a 2.5- to
4-day bulk MLR culture period. Control cultures were incubated in
the absence of hCTLA4-Ig, but were otherwise treated identically
to the hCTLA4-Ig-treated cultures. At the end of the culture period,
the bulk population was washed three times with medium to remove
any nonviable cells, resuspended, and added at a 1:l ratio to antiThyl.2 plus C’-treated BM. For experiments using primed splenocytes, hCTLA4-Ig (SO pg/mL final concentration), and/or anti-LFAl
MoAb ( 1 50 pglmL final concentration), or an equal volume of PBS
were added to the BMS preparation for 3 hours at 37°C. The BMS
preparation was infused without additional manipulation. All recipients received 25 X 10‘ anti-Thy1.2 plus C’-treated BM cells along
with 25 X 10‘ manipulated naive or primed splenocytes or nonmanipulated fresh splenocytes via caudal vein injection in 0.5 mL v01ume on the day after lethal irradiation.
Pathologic examination of tissues. Mice were killed, autopsied,
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2609
CTLA4 Ig and LFAl COBLOCKADE IN MURINE GVHD
and tissues were taken for histopathologic analysis. All samples
were placed in 10% neutral-buffered formalin, imbedded in paraffin,
sectioned, and stained with hematoxylin and eosin for histopathologic assessment. Organs were scored positive for GVHD if there
was single cell necrosis (skin, colon), crypt dropout (colon), periportal infiltrate with acute necrosis (liver), or endothelialitis with a
lymphocytic infiltrate ( l ~ n g ) . ~ ’In* ~previous
*
studies, these features
were present only in mice with active GVHD and not in irradiated
recipients of either syngeneic or anti-Thyl.2 plus C’-treated allogeneic BM.
Immunohistochemistry. Tissues (spleen, liver, and colon) were
embedded in OCT compound (Miles, Inc, Elkhart, IN), snap frozen
in liquid nitrogen and stored at -80°C. Serial 4-mm sections were
cut (IEC Minotome), thaw mounted onto glass slides, and fixedfor 5
minutes in acetone. After blocking with normal horse serum, sections
were incubated with biotinylated MoAbs specific for the following
molecules: B7-1 (clone 1G10, rat IgGZa, PharMingen, San Diego,
CA), B7-2 (clone GLl, rat IgG2a, PharMingen), ICAM-l (clone
Be2961, rat IgG2a, provided by Dr Adrienne Brian, University of
California San Diego)33and ICAM-2 (clone 3C4, rat IgG2a, PharMingen). Detection with peroxidase-conjugated avidin-biotin complex and 3,3’-diaminobenzadine as chromogen was performed essentially as describedM with reagents purchased from Vector
Laboratories, Inc (Burlingame, CA). The frequency of immunoperoxidase positive cells in tissue sections was quantitated using standard point counting technique as previously described35 and expressed as the number of positive staining cells per square millimeter
averaged in at least seven representative fields.
Analysis of splenocyte proliferationin response to host and thirdparty alloantigens. After in vitro culture, bulk MLR cell populations were washed, resuspended, and l@ cells plated into 96-well
round-bottom (Costar) plates. Tritiated thymidine (1 pCi) was added
16 hours before harvesting and counting, in the presence of scintillation fluid, on a beta counter. Freshly obtained or in vitro cultured
splenocyte preparations were subjected to limiting dilution analysis
(LDA) to estimate the precursor frequency of proliferating T lymphocytes (pPTL) of post-BMT splenocytes to host and third-party
alloantigens. Splenocytes were placed in 96-well flat round plates
(Costar) in threefold dilutions ranging from 1.5 X lo5 to 5 X 10’
cells per well in media plus sera as described above for bulk MLR
cultures. A fixed number (5 X l@) of irradiated (30 Cy), anti-Thyl.2
plus C’-treated host or third party (bm12) was added along with 10
U/mL recombinant IL-2 (Hoffman LaRoche, Nutley, NJ) in a final
volume of 200 pL/well. The plates were pulsed with tritiated thymidine after 7 days of incubation and harvested the next day. Using
Poisson distribution statistics according to the method of Taswell,”
the likelihood of a single hit was confirmed and a frequency estimate
calculated. Confidence intervals were obtained based upon the chisquare minimization and maximum likelihood methods to compare
the frequencies between the groups.
Flow cytometry analysis. Monoclonal antibodies (MoAbs) were
directly labeled with biotin, fluorescein isothiocyanate (FITC), or
phycoerythrin (PE) as previously described.” Single-cell splenocyte
preparations were suspended in buffer (PBS plus 5% colostrum-free
bovine serum + 0.015% sodium azide). Pelleted cells were incubated
for 15 minutes at 4°C with 0.4 pg of an anti-Fc receptor MoAb
(clone 2.462, provided by Dr Jay Unkless, Rockefeller University,
New York, NY) to prevent Fc binding.38Optimal concentrations of
directly conjugated (biotin-, PE-,and FITC-labeled) MoAbs were
added to a total volume of 100 to 130 pL and incubated for 1 hour
at4°C. For the biotin-labeled MoAb, fluorescence was indirectly
measured by adding streptavidin-labeled Red 613 (GIBCO) for an
additional hour at 4°C. After final washing, cells were fixed in 1%
paraformaldehyde.
For analysis ofin vitro cultured bulk population cells, one- or
two-color flow cytometry was performed using the following MoAbs
(obtained, unless otherwise indicated, from PharMingen): anti-CD3e
(clone 145-2C11, hamster IgG), anti-CD8 (clone 53-6.72, rat IgG2a.
provided by Dr Jeffrey Ledbetter, Bristol-Myers-Squibb, Seattle,
WA),39 anti-CD4 (clone GK1.5, rat IgG2b), anti-CD45R (B220)
(clone Ra3-6B2, rat IgG2a), anti-B7-l (clone 16.10A1, hamster
IgG),” antiCD28 (clone 37.5 1, hamster IgG), and an irrelevant rat
IgG2 antihuman antibody (3AlE):’
For analysis of donor or host origin of repopulating cells postBMT, two- or three-color flow cytometry was performed. Splenocytes were costained with anti-H-2b-SA-PE (clone EH144, mouse
IgG, provided by Dr T.V. Rajan, Albert Einstein University, New
York, NY) and anti-H-2’-FITC (clone 11-4.1, mouse IgG2a, American Type Culture Collection, Rockville, MD) after resuspending
single cell suspensions in buffer. The following antibodies and reagents were also used in conjunction witha11ti-H-2~ or anti-H-2k
MoAbs: anti-CD3e, anti-CD8, anti-CD4, B220, Mac1 alpha subunit
(CD18, clone MU70.15.11, rat IgG2b; PharMingen), and an irrelevant rat IgG2 antihuman antibody (3AlE).
All samples were analyzed on a FACScan (Becton Dickinson,
Mountain View, CA) using consort-30 software. A minimum of
20,000 events were examined. Background subtraction using a directly conjugated irrelevant antibody control was performed for each
sample.
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 ~hi-square.~’
Actuarial survival rates (the proportion of mice surviving on each day
post-BMT) are shown. P values < .05 were considered significant
and P values < .l, but > .05 were considered tobe a statistical
trend.
RESULTS
In vitro hCTLACIg addition to CS7BU6anti-B1O.BRbulk
cultured splenocytes inhibits the in vitro primary MLR, but
does not lead to a signijcantly reduced anti-GVHD effect
when the treated cells are infused in vivo. The proportion
of CD4+ or CD8’ T cells in the splenocyte population used
to initiate the bulk MLR culture was maintained at the end
of the culture period ( C M + T cells, 20% v 21%; CD8+ T
cells, 14% v 16%, respectively, for initiation v end of bulk
MLR). However, the proportion of T cells coexpressing the
IL-2R or B7-1 ligand substantially increased (IL-2R, 3% v
12%; B7-1, 3% v 9%, respectively, for initiation v end of
bulk MLR), consistent with donor splenocyte priming. We
have consistently observed an increase in the proportion of
cells at the endofthe
primary MLR
IL-2RtandB7-1+
reaction in this strain combination. Because B7-2is constitutively expressed at low levels on splenocytes and B7-1 expression is inducible on professionalAPCS;~wetested
whether the addition of hCTLA4-Ig (to block B7 binding to
CD28lCTLA4) atthe initiation of bulk culture would reduce
T-cell activation as measured by proliferationandIL-2R
expression on T cells. Initial studies were performed to determine the optimal concentration (5, 20, 25, or 50 pg/mL) of
hCTLA4-Ig necessary to inhibit the primary MLR response
of C57BLJ6 anti-B10.BR bulk cultured splenocytes. In these
experiments, as well as others involving different MHC disparate donor antihost strain combinations, maximum inhibition assessed o n the day of peak proliferation of the control
cultures was consistently observed at concentrations of 2
20 pg/mL(datanotshown).Therefore,inallsubsequent
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BLAZAR ET AL
2610
Table 1. Addition of hCTLA4-Ig Protein t o Allogeneic Stimulator
Cells Decreases In Vitro Primary MLR Alloresponses
Vitro
In
18
Treatment
Experiment 1
Sham incubated
33.1
hCTLA-Ig6.1
50 pg/mL
Experiment 2
100 Sham incubated
40.4
hCTLA-Ig 13.1
50 pg/mL
% Recovery
A cpm
63
56
% Control
cpm
100
L
>
a
3
v)
63
54
31
C57BU6 splenocytes (8 x 106/mL) were incubated with irradiated
anti-Thyl.2 + C'-treated B1O.BR stimulators (1:l ratio)in the presence
or absence of hCTLA4-Ig (50 pg/mL). The percentage of recovery is
number of responders remaining divided by input cell
number x
100%. Splenocytes were maintained for 3 days, plated at lo5cell per
well. and pulsed overnight. A cpm ~cPms.p.,im..t.l
- cpm,..wnd.r.alons)
Standard
are listed as mean values for 12 wells per group x
deviations were 515% of mean values.
_.",
Table 2. Coblockade of the LFA1:ICAM Pathway in Context of
hCTLAClg Protein Further Reduces Alloresponsiveness In Vitro
In Vitro Treatment
Experiment 1
Sham incubated
hCTLA4-Ig
Anti-LFAl
hCTLA4-Ig + anti-LFAl
Experiment 2
Sham incubated
hCTLA4-Ig
Anti-LFAl
hCTLA4-Ig + anti-LFAl
Experiment 3
Sham incubated
hCTLA4-Ig
hCTLA4-la anti-LFAl
+
A cpm
% Control cpm
43'
38'
37 '
25'
36.8
17.1
6.2
0.0
100
46
17
52
38
38
27
39.0
12.3
4.8
0.9
100
32
14
2
116
55
37
14.2
3.8
1.6
100
27
11
% Recovery
0
C57BU6 splenocytes (8 x 106/mL) were incubated with irradiated
B1O.BR stimulators (1:l ratio)in the presence or absence of hCTLA4lg (50 pg/mL) alone, anti-LFAl antibody (150 pglmL), or hCTLA4-Ig
plus anti-LFAl antibody. The percentage recovery is number of responders remaining divided by input cell number x 100%. Splenocytes were maintained for 4 days, plated at lo5 cell per well, and
pulsed overnight. A cpm (cpmexw,imen,al- cpm,..wnondenalons)
are listed
Standard deviations
as mean values for 12 wells per group x
were 515% of mean values.
'Recoveries in experiment 1 were determined after Ficoll-Hypaque
density gradient centrifugation and washing.
,
.
10
I
20
.
I
-
.
.
I
30
40
-
I
50
DAYS POST-BMT
B ' O
experiments, the highest concentration tested (50 pg/mL) of
hCTLA4-lg was used.
The results offive representative experiments in which
hCTLA4-Ig (50 pg/mL) was added to a C57BL/6 antiBIO.BR bulk MLR culture are summarized in Tables 1 and
2. As compared with the sham-treated control cultures, responder cells were induced into a state of hyporesponsiveness by hCTLA4-Ig blockade of irradiated fully allogeneic stimulators (54% to 82% reduction) and led to cell
recoveries ranging from 38% to 55% of input cell number.
To determine if the reductions in donor antihost proliferative responses were sufficient for GVHD protection in vivo,
.
0
f
.*
Y
m
.
..
0
BM (No Sploon)
Fig 1. The effect on in vivo GVHD-inducing capacity by inhibiting
of CD28/CTLA4B7 alone or in conjunction with
thesignaling
LFA1:ICAM in donor splenocytes during exposure t o host alloantigens in vitro: C57BL/6 splenocytes (8 x 106/mL) were cultured with
irradiated B1O.BR splenocytes (8 x 106/mL)for 3 days in the presence
or absence of hCTLA44g protein (50 pg/mL) alone or hCTlA4-Ig plus
anti-LFAl (150 pg/mL) antibody, as indicated. Cells were then
washed extensively, and 25 x 10' splenocytes were infused along
with 25 x lo6 C57BL/6 anti-Thyl.2 plus C-treated BM into lethally
irradiated B1O.BR recipients. Eight miceper group weretransplanted.
Actuarial survival is plotted.
incubated cells from the two groups in experiment 2 (Table
1 ) were extensively washed to remove all detectable nonviable cells, resuspended, and 25 X 10' cells reinfused along
with 25 X 10' anti-Thy1.2 plus C' treated, freshly harvested
donor BM into BIO.BR recipients (n = 8 mice/group). Flow
cytometry analysis of donor splenocytes that were sham cultured with irradiated stimulator cells or cultured in the presence of hCTLA4-Ig-treated showed highly similar proportions ofCD4' (12% v 14%, respectively) or CD8' (9% v
8%, respectively) just before in vivo infusion. The actuarial
survival rate is shownin Fig I . In vitro (3-day) donor antihost
stimulated cells were sufficiently alloreactive to result in
uniform lethality within the first 5 weeks post-BMT. As
compared with the recipients of sham-treated inocula, recipients of either hCTLA4-Ig-treated stimulators had a marginallyprolonged ( P = .057) actuarial survival rate in vivo,
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CTLA4 lg and LFAl COBLOCKADE IN MURINEGVHD
although none of the mice that received the hCTLA4-Igtreated inocula survived the observation period. Examination
of the post-BMT weight curves did not show any obvious
benefit of infusing the treated as compared with the control
cell populations. At no time period post-BMT did the mean
weight curves of recipients of treated inocula appear to be
superior to the sham-incubated control group (data not
shown). The marginal actuarial survival rate prolongation
conferred by infusing donor splenocytes exposed to
hCTLA4-Ig-treated stimulator cells, as compared with the
control group (Fig 1) was found not to be reproducible when
the treated cells from experiment 3 (Table 2) were infused in
vivo (data not shown). When data from these two identically
performed experiments were pooled for analysis, there was
no significant difference noted in actuarial survival rates
when comparing recipients of sham-incubated donor splenocytes to recipients of hCTLA4-Ig-treated splenocytes, with
the latter group having an actuarial survival rate that was
not significantly (P= .5 1, respectively) higher than the control group. We concluded from these experiments that the
reduction in the proliferative responses observed in
hCTLA4-Ig-treated cultures was insufficient to provide any
reproducible evidence of protection from GVHD-induced
mortality in vivo.
Coblockade of CD28/CTLA4:B7 and LFA1:ICAM pathways in naive donor splenocytes leads to a variable and
incomplete reduction of GVHD in lethally irradiated recipients of fully allogeneic donor grafts. From the in vitro
experiments discussed above as well as our previous studies
in which hCTLA4-Ig was infused in vivo as a means of
preventing GVHD, we concluded that in vitro blockade of
the CD28/CTLA4:B7 pathway alone would be inadequate
in this GVHD model system. One possible explanation for
these findings is that other T cel1:APC pathways capable
of providing a T-cell signal could overcome the partially
inhibitory effects of CD28/CTLA4:B7 blockade. One such
candidate interaction could be the LFA1:ICAM pathway.
We focused on this pathway in part because of studies by
other investigators who have shown that the in vivo blockade
of LFA1:ICAM interaction facilitates the acceptance of solid
organ grafts and results in a diminished GVHD response in
mice. To determine if B7 ligands and ICAM counterreceptors were potentially involved in GVHD generation in vivo,
we embarked upon immunohistologic analysis of tissues obtained from lethally irradiated B1O.BR recipients of 25 X
IO6 anti-Thy1.2 plus C'-treated BM with 25 X lo6nontreated
(GVHD positive control) or anti-Thy 1.2 plus C'-treated
splenocytes (GVHD-negative, BMT control). The frequency
of B7-I, B7-2, ICAM-1, or ICAM-2 expressing cells in
spleen, colon, and liver of recipients of nondepleted or antiThy 1.2 plus C'-treated supplemental splenocytes is shown
in Table 3. These data indicate that the graft-versus-host
reaction in this model system is associated with the upregulation of B7-1, B7-2, andlor ICAM-1 in all three tissues, suggesting that these ligands may be involved in GVHD generation
in
vivo. Figure 2 is a photomicrograph of the
immunohistologic results of B7 and ICAM ligand upregulation in representative splenic sections taken from recipients
of anti-Thyl.2 plus C'-treated (Fig 2A) or untreated (Fig
2B) donor splenocytes.
2611
Table 3. Quantitative Tissue In S i Immunohistologic Assessment
of Cell Sudace Expressionof B7 and C A M Ligands DuringGVHD
In Vitro Treatment
Tissue
(anti-Thyl.2+ C')"
Spleen
No
Yes
No
Yes
No
Yes
Colon
Liver
87-1
87-2
ICAM-l
329 296
0
5
0
85
45
0
310
150
243
37
317
0
43
0
9
0
35
2
CAM-2
306
56
80
24
Frozen-tissue sections were obtained from B1O.BR recipients of
C57BU6 donor BMand splenocytes on day 14 post-BMT. In situ immunoperoxidase staining of the indicated tissues were performed and
the proportion of cells expressing the indicated ligands were quantitated as described in Materials and Methods. Thedata shown are
expressed as the number of positive cells/mm*.
In vitro treatment (anti-Thyl.2 + C') refers to whether or not the
donor splenocytes were treated in vitro with anti-Thyl.2 + C' to remove GVHD-causing cells.
Based upon our in vivo GVHD immunohistologic data in
this model system, we proceeded to experiments designed
to coblock both the CD28:B7-1 and LFA1:ICAM pathways
in vitro. The results of three primary MLR experiments in
which the effect of hCTLA4-Ig andlor anti-LFAl antibody
blockade of bulk MLR cultured cells was tested are shown
in Tables 2 and 4. Both hCTLA4-Ig and anti-LFAl antibody
alone were inhibitory to the C57BL/6 anti-B 10.BR proliferative response. The combination of hCTLA4-Ig and antiLFAl provided the highest degree of inhibition, resulting in
only a 0% to 11% of control response. Cells from experiment
1 (Table 2) were isolated by Ficoll-Hypaque density-gradient
centrifugation and the bulk population subjected to flow cytometry analysis. These data show that hCTLA4-Ig and antiLFAl antibody can reduce the expansion of CD8+ and IL2R+ cells from 19% to 2% in this MLR culture (Table 4).
LDA analysis of C57BU6 responders that are nonmanipulated or depleted of either CD4+ or CD8+ T cells before
culturing withBlO.BR stimulators has shown that CD8+
cells account for a majority of the proliferative responses
(data not shown). These results are both consistent with early
(day 5 through 7 post-BMT) studies in this model system
that have shown a predominance of CD8+ T cells as GVHD
effector cells as detected by in situ immunoperoxidase staining of GVHD target tissues (lung, liver, and colon) and
flow cytometry analysis of cells obtained by thoracic duct
cannulation (data not shown).
In each experiment, 225% of the number of input cells
was recovered, which permitted testing of the anti-GVHD
effect of hCTLA4-Ig plus anti-LFAl in vivo. C57BL/6 antiB 10.BR in vitro bulk-cultured cells incubated under control
conditions or in the presence of hCTLA4-Ig alone or
hCTLA4-Ig plus anti-LFAl antibody were washed and 25
X lo6 supplemental cultured splenocytes infused in vivo
along with anti-Thyl.2 plus C'-treated BM.As compared
with sham incubated control cells, neither hCTLA4-Ig alone
nor hCTLA4-Ig plus anti-LFAl antibody treatment provided
any evidence of GVHD protection by actuarial survival rates
or mean weight data (data not shown). The absence of any
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BLAZAR ET AL
2612
A
Fig 2. Upregulation of B7 and C A M ligands in the spleen obtained from mice undergoing graft-versus-host
a
reaction. lmmunoperoxidase
staining for B7 and ICAM ligands is shownin representative splenic sections obtained from BIO.BR recipients of anti-Thyl.2 plus C'-treated
C57BL/6 BM and anti-Thyl.2 plus C'-treated (A) or nontreated (B) C57BL/6 splenocytes on day 14 post-BMT. As compared with the controls
(Al. there is an increase in the proportion of splenocytes expressing 87-1, 87-2, ICAM-1, and ICAM-2 ligands in mice that have histologically
documented GVHD in the colon andliver. The frequency of cells expressing these ligands in the spleen, colon, and liver are listed in Table 3.
obvious decrement in GVHD capacity was observed despite
the 89% inhibition of donor antihost proliferation observed
when an aliquot of these same cells was analyzed in vitro
(Table 2, experiment 3) and despite infusing a lower proportion of T cells (CD4' or CD8') that coexpress IL-2R (Table
4). These data indicate that combination hCTLA4-Ig + antiLFA I treatment in vitro was entirely ineffective in reducing
GVHD.
Coblockode of CD28/CTLA4:B7 and LFA1:ICAM pathways in donor antihost primed splenocytes lead.7 to a significant reduction of GVHD in lethallv irradiated recipients
q f j d l v allogeneic donor grajis. At this juncture, we considered the possibility that naive murine splenocytes would
bemore difficult than primed cells to induce into a state
of sufficient antigen-specific hyporesponsiveness. A large
proportion of the published murine literature has examined
anergy induction of antigen-specific and hence primed Tcell clones. Moreover, Turka et al" have shown that whereas
hCTLA4-Ig-treated rat recipients of cardiac allografts maintained their graft rejection capacity, rats first primed in vivo
Table 4. Effect of Coblockade of the LFA1:ICAM Pathway in the
Context of hCTLA4-1g Protein T-, B-, and NK Cell
Expansion and Activation In Vitro
%IL-ZR'
CD4' or
In VitroTreatment
10
Sham incubated
59
5
hCTLA4-Ig
Anti-LFAl
4
hCTLA4-Ig
+ anti-LFAl
%CD4'
%CDB'
19
8
CD8'
%B220'
16
71
9
SbNK1.1'
5
7
14
6
67
5
217
6
64
6
Cells from experiment 1, Table 2 were phenotyped after the 4-day
MLR culture as described in Materials and Methods. Listed are the
percentages of positive cells in these bulk cultures.
Abbreviation: NK, natural killer.
with irradiated donor leukocytes and then subsequently given
hCTLA4-Ig achieved permanent and specific cardiac allograft acceptance. Therefore, rather than to make naive donor
splenocytes tolerant, we examined the effects ofCD281
CTLA4:B7 and/or LFAIKAM in vitro blockade on inhibiting the GVHD-induced lethality of C57BL/6 anti-B10.BR
bulk-cultured splenocytes. At theend of a 3-daypriming
period, hCTLA4-lg protein and/or anti-LFAI antibody was
added to the cultured cells for 3 hourstopermitligand
saturation, the cells washed. and 25 X 10' control or treated
splenocytes infused in vivo along with anti-Thy 1.2 plus C'treated freshly harvested BM. An aliquot of the bulk cultured
cells was analyzed for tritiated thymidine incorporation after
an additional 24- or 48-hour incubation period. which documented their proliferative capacity (counts per
minute
[cpm] sllopencir rc.ipont~en. aulo~oCoarh g c k g n d = 12.0 and 14.9 x 10'
cpm, respectively). The proportion of IL-2R' cells in these
cultures was 2796, consistent with the functional data and
indicating that alloreactivity had taken place.
Becausethe3-hourexposureperiodwouldlikely
be too
short to fully induce antigen-specific hyporesponsiveness~.".JJ
and because B7 and ICAM ligands are upregulated during
GVHD, we continued the same reagents in vivo for 28 days
post-BMT to complete the hyporesponsiveness induction
process. Our previous experiments indicated that the in vivo
combination of hCTLA4-Ig plus anti-LFA 1 alone was ineffective in preventing lethal GVHD (data not shown). The
actuarial survival results of the current studies are depicted
in Fig 3A and the mean weight curves in Fig 3B. As compared with the control group, recipients receiving donor antihost primed splenocytes exposed to hCTLA4-lg in vitro and
in vivo had a marginally ( P = . l ) higher actuarial survival
ratewith12%
(v 0% of controls) surviving the138-day
observation period. In contrast, 88% of the recipients of
hCTLA4-lg plus anti-LFAI survived, a significantly higher
proportion than either the PBS (P < .0002) or hCTLA4-lg
only (P = .001) groups. Mice receiving hCTLA4-lg plus
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CTLA4 lg and LFAl COBLOCKADE IN MURINEGVHD
pas
,
.
0.0
20
0
60
40
80
l00
120
140
l11
138
DAYS POST-BM1
2613
cpm
. ,,,,"
h:lckpn d = 16.6 X IO', assessed after
pulsing an aliquot of cells for 18 hours).
Control recipients of in vitro plus in vivo hCTLA4-Ig or
in vivo anti-LFA 1 alone had significantly ( P = .00025, .032,
respectively) higher actuarial survival rates as compared with
the PBS control group (Fig 4A). Both reagents were required
for the optimal biologic effect in this experiment because
the addition of hCTLA4-Ig to anti-LFA 1 in vitro and in vivo
resulted in a significantly higher actuarial survival rate in
these mice as compared with those receiving only anti-LFAl
or hCTLA4-Ig given in vitro plus in vivo ( P = .033, .OOS I ,
respectively) (Fig 4A). Whereas the actuarial survival rates
of recipients of hCTLA4-Ig plus anti-LFAI given in vitro
and in vivo were 100%, most of the mice were not
GVHD
free. Mean weight in this group averaged 90% of pre-RMT
weight values (Fig 4B). The mean weight and immunologic
data (see below) are indicative of a mild, sublethal GVHD
process. Taken together, these data indicate that there is an
A ' O
12
!z
.
.
10
.
a
30
58
78
91
I'.."
DAYS POST-BUT
Fig 3. hCTLAClg plus anti-LFAl in vitro treatment of donor antihost primedsplenocytes followed byin vivo administration of these
same reagents markedly reduces the GVHD capacity of the donor
inocula: C57BL/6 splenocytes (8 x 106/mL) were culturedwith irradiated B1O.BR splenocytes (8 x 10E/mL)for 3 days. After the priming
period, cells were exposed t o saline, hCTLA4-Ig protein (50 pg/mLl
alone or hCTLA4-Ig plus anti-LFAl (150 pg/mL) antibody, as indicated, for a period of 3 hours on ice t o permit receptor saturation.
After the additional culture period, cells were washed extensively,
and 25 x lo6 splenocytes were infused along with 25 x lo6 C57BL/6
anti-Thyl.2 plus C-treated BM into lethally irradiated B1O.BR recipients. Post-BMT, recipients were injected in vivo with the same reagents used for the particular
in vitro treatmentused for thatgroup.
Recipients were treated with PBS and hCTLA4-Ig (250 p g on days
-1 and 0, and 100 p g thrice weekly through day 28 post-BMT) with
or without anti-LFAl antibody (300 p g twice weekly on days "-1
through day 29 post-BMTI as indicated. Eight mice per group were
transplanted. (A) Actuarial survival is plotted;(B) mean weight data
for the same experiment are plotted.
anti-LFAI treatment had mean weight values that exceeded
the pre-BMT bodyweightat
the end of the observation
period (Fig 3B), consistent with their healthy appearance
and lack of clinical evidence of GVHD. These mean weight
values were significantly higher than the PBS control group
beginning on day 20 post-BMT.
To determine whether the anti-GVHD effect of hCTLA4Ig plus anti-LFAI was reproducible and to sort outwhat
themore important components of our approach are, we
performed a second experiment according to the protocol
shown in Fig 3 and included a number of additional control
groups. These additional groups consisted of mice receiving
anti-LFAI antibody alone in vitro and in vivo, anti-LFAI
antibody plus hCTLA4-lg together in vivo alone, or no cultured splenocytes. C57BW6 anti-B IO.BR-primed splenocytes had a comparable proliferative indexto those cells
usedto infuse mice in Fig 3 (in the current experiment,
i.............
."...."...."..
I
O
0.0 0
B
.
n 2 0 llqroup
2
40
DAYS
POST-BUT
EO
L
10
9+
hCtU4-l
a l00
anll-LCAl In VIVO
mll-LFAl
antl-LFAl
In vllm md
In VIVO
120S
"1
Fig 4. hCTLAClg plus anti-LFAl in v-ho treatment of donor antihost primedsplenocytes followed byin vivo administration ofthese
same reagents markedly reduces the GVHD capacity of the donor
inocula: C57BL16 splenocytes (8 x 106/mL)were culturedwith irradiated B1O.BR splenocytes (8 x 1OE/rnL)for 3 days. After the priming
period, cells were exposed t o saline, hCTLA4-Ig protein (50 pg/mL)
and/or anti-LFAl (150 pg/mLl antibody, as indicated, for a period of
3 hours on ice t o permit receptor saturation. After the additional
culture period, cells were washed extensively, and 25 x lo6 splenocytes were infused along with 25 x lo6 C57BL/6 anti-Thyl.2 plus
C'-treated BM into lethally irradiated B1O.BR recipients. Post-BMT,
recipients were injectedin vivo with thesame reagents used for the
particular in vitro treatment used for that group. Recipients were
treated with PBS, hCTLA4-Ig (250 p g on days -1 and 0, and 100 p g
thrice weekly throughday 28 post-BMTI, and/or anti-LFAl antibody
(300 p g twice weekly ondays - 1through day 29 post-BMT) as indicated. Eight miceper group weretransplanted. (A) Actuarial survival
is plotted;(B) mean weight data for thesame experiment are plotted.
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BLAZAR ET AL
2614
...-."
l34
.I
~~.
0
20
ab40
ab
.
.=..""."""
-a
PBS
,
l9
39
59
l9
99
l00
DAYS POST-BMT
DAYS POST-BUT
Fig 5. hCTLA4-lg plus anti-LFAlin vitro treatment of donor
splenocytes followed byin vivo administration of these
same reagents markedly
reduces the GVHD capacity of the donorinocula effect of priming beforein vitro treatment on
in vivo GVHD generation: C57BL/6 splenocytes
18 x 1O6/mL1were cultured with irradiated B1O.BR splenocytes (8 x 106/mL). To generate primed cells, a 3-day incubation period was used
prior t o a 3-hour in vitro treatment. For nonprimed cells, reagents were added on the day of culture initiation. In both instances, bulk MLR
cultures were exposed to saline, hCTLA4-Ig protein (50 pg/mL) and/or anti-LFAl 1150 pg/mL) antibody, as indicated, for a period of 3days
Inonprimed cells) or 3 hours (primedcells). After the additional cultureperiod, cells were washed extensively, and 25 x lo6 splenocytes were
infused along with 25 x lo6 C57BL/6 anti-Thyl.2 plus C'-treated BM into lethally irradiated B1O.BR recipients. Post-BMT, recipients were
injected in vivo with the same reagents used for the particular in vitro treatment used for that group. Recipients were treated with PBS,
hCTLA4-Ig (250p g o n
days -1 and 0, and 100 p g on thrice weekly through
day 28 post-BMTI, and/or anti-LFAl antibody1300 p g twice weekly
weretransplanted. (A) Actuarialsurvival is plotted; (B) mean weight
on days -1 through day 29 post-BMTI as indicated. Eiqht
- miceDer
. aroup
data for the same experiment are plotted.
apparent requirement for hCTLA4-Ig + anti-LFAI to be
added in vitro before in vivo infusion because recipients
receiving splenocytes exposed to these reagents in vitro and
in vivo had a statistical trend ( P = .07)toward a higher
actuarial survival rate as compared with recipients of nontreated splenocytes and hCTLA4-Ig plus anti-LFA1 given
in vivo (Fig 4A). Of greater importance, the combination of
hCTLA4-lg plus anti-LFA1 in vitro and in vivo did provide
a reproducible and significantly higher degree of protection
from GVHD-induced mortality confirming the data shown
in Fig 3A.
The in vitro and in vivo coblockade of CD28/CTLA4:B7
and LFA1:ICAM pathways i s more effective in preventing
GVHD when primed rather than when naive donor splenocytes are infused in vivo. To address whether recipients of
primed cells might experience a less potent anti-GVHD effect conferred by hCTLA4-lg plus anti-LFAI treatment
when compared with recipients of naive splenocytes, additional groups of mice in the previously described experiment
were given naive splenocytes that were sham-incubated or
incubated withhCTLA4-Ig plus anti-LFAI in vitro for 3
hours before in vivo infusion. Mice receiving primed or
naive cells incubated with hCTLA4-Ig plus anti-LFA1 also
received these reagents in vivo for 28 days post-BMT. Recipients of nonprimed cells and hCTLA4-Ig plus anti-LFAI had
a significantly ( P = .045)higher actuarial survival rate (Fig
5A) as compared with their respective PBS controls. Recipients of primed cells and in vitro and in vivo hCTLA4-Ig
plus anti-LFAI had a higher ( P = .074) survival rate than
those receiving nonprimed cells (100% v 75%. respectively)
(Fig 5A), suggesting that the use of primed cells offers an
advantage over naive cells, even though control recipients
of primed or nonprimed splenocytes had a comparable ( P =
.39) actuarial survival rate.
The in vitro and in vivo coblockade of CD28/CTLA4:B7
and LFA1:ICAM pathways does not lead to a prolonged
state of irnmunocompromiseas compared with recipients of
anti-Thyl.2 plus C'-treated BMS. We sought to ask
whether recipients of primed splenocytes exposed to
hCTLA4-lg plus anti-LFA1 in vitro and in vivo or in vivo
only were more immunosuppressed than recipients of antiThyl.2 plus C'-treated BM without supplemental splenocytes. Representative mice (mean weights were 2 20 g in
the three groups) were analyzed at 5 months post-BMT.
Despite the histologic evidence of mononuclear cell infiltration in some of these mice (data not shown), flow cytometry
analysis of the spleenat this time post-BMT showedno
evidence of hypocellularity, T- or B-cell lymphopenia, or
increases in the relative proportion of macrophages typically
associated with acute, active GVHD in this model system
(Table 5). Recipients that received hCTLA4-lg plus antiLFAl in vitro and/or in vivo hadno evidence of residual
host cells, in contrast with animals that did not receive supplemental splenocytes and either of these proteins. Two additionalmiceper
group thatreceived anti-Thy1.2 plus C'treated BM andno supplemental splenocytes or primed
splenocytes incubatedwith hCTLA4-lg plus anti-LFAI in
vitro followed by in vivo infusions of these reagents were
studied at 18 1 days post-BMT. By this time, in the recipients
of hCTLA4-Ig plus anti-LFA1 in vitro and in vivo, there
was evidence of a modest decrease in overall splenic cellularity and a modest T-cell lymphopenia. The B-cell and macrophage compartments appeared to be relatively spared from
the graft-versus-host reaction. Taken together, these data are
consistent with the clinical appearance of a nonlethal form
of GVHD (Table 5).
We concurrently pursued the issue as to whether the recipients of hCTLA4-lg plus anti-LFAI were globally immunosuppressed. When analyzed at 5 months post-BMT, splenic
pPTL analysis of donor antihost and anti-third party (bm 12)
alloreactive proliferating cells (Table 6) showed comparable
frequencies against both types of stimulator cells in each
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LFAl CTLA4 Ig and2615
COBLOCKADE
GVHD IN MURINE
+
Table 5. Effect of CD28/CTLA4 B7 LFA1:ICAM Blockade on Splenic Phenotypic Reconstitution in Long-term Post-BMT
Survivors Receiving In V i 0 Donor Antihost Primed Cells
Total no.
Group
Day 151 post-BMT
BM without splenocytes
hCTLA4-lg anti-LFAI in vitro in vivo
hCTLA4-lg anti-LFAl in vivo only
Day 181 post-BMT
BM without splenocytes
65.2
hCTLA4-Ig anti-LFAl in vitro in vivo
+
+
+
+
+
64.4
179.2
104.4
80.0
66.5
H-2‘
H-2b
H-Zb CD4
H-Zb CD8
10.9
5.2
1.3
57.3
166.7 14.3 0.0
9.4
0.0
96.0
41.2
120.1
25.1
77.312.5
5.2
16.1
6.3
14.4
74.4
6.0
42.6 4.7
8.0
4.1
1.6
0.0
H-Zb B220
H-2b Mac1
52.8
8.8
Spleens were obtained at 151 or 181 days post-BMT from two representative mice per group from Table 4 and Fig 4 were pooled together
at each time point before analysis by two-color flow cytometry. Values listed are absolute numbers of cells x lo-’. Details of the in vitro and
in vivo treatments are described in the legend to Fig 4.
of the three groups. Because C57BU6 anti-BlO.BR splenic
pPTL precursor frequencies in nontransplanted mice were
more than 13-fold higher on days 151 and 191 post-BMT,
mice in each group appeared to be hyporesponsive to host
alloantigens. In contrast, anti-third party (anti-bml2) responses in nontransplanted controls were less than 1.5-fold
higher than transplanted mice at both time periods in all
groups with the single exception of recipients of hCTLA4Ig plus anti-LFAl on day 181. The latter data could be
indicative of GVHD-induced immunosuppression, consistent with the weight data and splenic flow cytometry data
and indicative of a graft-versus-host reaction.
DISCUSSION
The major finding of this study is that B7 and ICAM
ligands are upregulated during GVHD generation and the
combination of hCTLA4-Ig plus anti-LFAl antibody can
significantly and reproducibly decrease acute GVHD-induced in lethally irradiated recipients of fully allogeneic donor grafts. In addition, we have shown that the in vitro
exposure of donor splenocytes to host alloantigens under
conditions in which CD28/CTLA4:B7 signaling is limited
does not eliminate GVHD induced across MHC class I, 11,
and miH disparities. These are the first in vivo GVHD studies
that directly test the theory that the in vitro blockade of
the CD28/CTLA4:B7 pathway alone or in combination with
coblockade of the LFA1:ICAM T-cell costimulatory pathway would be. sufficient for GVHD prevention in mice.
One of the goals of studies such as ours is to develop
approaches that are encouraging for clinical applications.
Theobald et a14’ have shown that the frequency of IL-2producing T cells (precursor helper T lymphocytes [pHTL])
correlates with the incidence of grades 11-IV GVHD in recipients of histocompatible sibling donor grafts. Whereas we
do not know in the strain combination used in this study
whether an in vitro MLR response or pFTL frequency would
be predictive of the incidence andor severity of GVHD,
reducing the pHTL frequency of donor antihost alloreactive
cells by CD28/CTLA4:B7 and LFA1:ICAM coblockade in
vitro and/or in vivo could be exploited as a means of GVHD
prevention in humans. Tan et a l l ’ showed that the CD4+ T
cell MLR response to fully allogeneic stimulators was reduced by 50% to 85%, consistent with our murine bulk MLR
cultures. Similar primary MLR results have been reported
by Gribben et a1,46 who also showed that pHTL responses
between histoincompatible donors and recipients could be
reduced up to 1,000-fold. These data would imply that
hCTLA4-Ig inhibition of donor antihost alloreactive cells in
Table 6. Effect of CD28/CTLA4B7 + LFA1:ICAM Blockade on Splenic Antihost and Anti-Third Party pPTL Frequencies in Long-term
Post-BMT Survivors Receiving In Vitro Donor Antihost Primed Cells
Group
Day 151 post-BMT
BM without splenocytes
hCTLAGlg/anti-LFAl
hCTLA4-lg/anti-LFAl
Nontransplanted B6
Day 181 post-BMT
BM without splenocytes
hCTLAClg/anti-LFAl
Nontransplanted B6
In Vitro Tx
None
Yes
None
NA
None
Yes
NA
Vivo
In
Tx
PBS
1/5,913
Yes
117,165
1/4,761
Yes
NA
1/3,696
PBS
Yes
NA
Anti-B1O.BR
-
Anti-bml2
1t17.964
1/18,654
1/20,638
1t1.234
V19.842
V22.653
111.364
1t8.198
V31.780
V6.310
Spleens were obtained at 151 days post-BMT from two representative mice per group shown in Fig 4 and Table 4 were pooled at each time
point and analyzed in an LDA for determination of antihost (B1O.BR) and anti-third party (anti-bml2) pPTL precursor frequencies. In addition,
nontransplanted C57BU6 (66) control micewere used on each assay day for pPTL precursor frequencies. Responder cells were incubated for
7 days in the presence of T-cell-depleted irradiated stimulators and IL-2 (10 U/mL) in mcirotiter wells. Cells were labeled overnight with tritiated
thymidine, harvested, and counted. All frequencies conformed to the Poisson distribution.
Abbreviations: Tx, treatment: NA, not applicable.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2616
vitro might be effective in reducing GVHD. However, our
results would suggest that the anti-GVHD effect
of hCTLA4Ig in vitromight be incomplete. The incomplete effectis
not likely caused by suboptimal CD28/CTLA4:B7 blockade
because (1) hCTLA4-Ig was used at concentrations severalfold higher than necessary to achieve optimal MLR inhibition; (2)concurrentexperiments
withparaformaldehydefixation (0.15% or 0.5%) ofhost splenocytes provideda
similar MLR inhibition (n = 3 experiments)and in vivo
GVHD effect (n = 2 experiments) as hCTLA4-lg treatment
Paraformaldedyde-fixation precludes
of hostsplenocytes.
cellsurface upregulation of ligands generated during an
MLR so that B7-1 is nondetectable on the host A P C S ~at~
the end of the culture period and only the low level of B72constitutivelyexpressed
on hostAPCs”couldbind
to
CD28/CTLA4 on the donor splenic T cells.
At this point,weconcludedthatthedonor
antihost
GVHD-causing T cells may have been partially resistant to
CD28/CTLA4:B7blockadebecause of theparticular cell
of the prestype involved in the GVHD response or because
ence of T-cell-associated determinants capableof providing
costimulation. It is possible that use of another strain combination for MLR and GVHD generation might be more susceptible to hCTLA4-Igblockade. For example, becauseThl
(IL-2-producing) cells appear to be dependent upon CD28/
CTLA4:B7 signaling, perhapsin contrast with IL-4-producing Th2 cell^,"^'"^^ it will be important to test this approach
in a GVHD system in which only T h l cells are required for
in vitro alloresponse and lethality in V ~ V O . ~ ”We
~ ~ ’focused
on the coblockade T cell LFAl and APC ICAM interaction
because studies by other investigators have shown that signaling through the LFA1:ICAM pathway can costimulate T
cells. Damle eta1” have shownthat LFAl :ICAM and CD28/
CTLA4:B7pathways synergistically activateeither nonprimed or primed antigen-specific CD4’ T cells, suggesting
that the coblockade of these pathways may be highly desirable for efficiently inhibiting donor antihost alloresponses.
Greenet als4 concludedfrom aseries of experiments in
which the TCR is triggered by staphylococcal enterotoxins,
immobilized anti-CD3 epsilon antibody, or phorbol esters,
that LFAI, in contrast with CD28, does not function as a
costimulatory molecule, but serves primarily to modulate the
signal delivered through the TCR. As related to our studies,
the use of hCTLA4-Igandanti-LFAI
antibodytogether
would modify signals transduced via the CD28/CTLA4:B7
and the TCR pathways that could be additive or synergistic
in shutting down alloresponses. Both our in vitro and in vivo
data support this hypothesis.
Our mostpromisingresultswith
hCTLA4-Ig plusantiLFAl usedprimedrather
than naive cellsandcontinued
infusion of these reagents in vivo. With this combined approach, acute GVHD-induced lethality has been eliminated
and the majority, but not all, of long-term post-BMT chimeras are clinically healthy without evidence of GVHD. Why
some T cells may have escaped this combined blockade is
not known. Perhaps the T-cell costimulatory properties of
one of the alternative 1igand:receptor interactions discussed
earlier was sufficient to partially compensate for the potent
inhibitory effects of hCTLA4-Ig plus anti-LFA I . In that re-
BLAZAR ET AL
gard, the mechanisms involvedin the beneficial anti-GVHD
effects observed withthisapproach
arealso unknown.A
prolonged in vitro incubation period would
be required to
show specificity of inhibition of alloresponsiveness
with a
minimum of 3 daysfor priming, 2 to 4 daysfor anergy
induction, and 3 to 5 days for assessment of antihost and
anti-third party responses (total 2 8 to13days).
lnitial
experiments to assess whether antihost-specific hyporesponsiveness has been obtained have been uninterpretable,
and
additional studies will be needed to elucidate the exact
mechanisms involved.
A potential drawback is that the in vivo administration of
hCTLA4-Ig andor anti-LFA1 antibody could result in a state
of prolonged immunosuppression in the recipient. However,
we elected to administerthese reagents in vivo after incubating the primed donor splenocytes in vitro for 2 reasons: (1)
the kinetics of antigen-specifichyporesponsivenessinduction have been estimated to require at least 1 to 2 days for
full biologic effects. We have not as yet been able to test
how long primed splenocytes will retain their GVHD capacity beyond the 4-day culture period we have used for these
studies. (2) We have observed the upregulation of B7-1 and
B7-2 ligands andICAM-I in vivo during theearlypostBMT GVHD response in this strain combination. Thus, if
donor antihost alloresponsiveness is not completely achieved
in vitro, it is possible that costimulation of the small proportion of donor cells that have escaped anergyinduction in
vitro could be sufficient for inducing GVHD lethality.
As assessed by phenotype and function, we could find no
indication that hCTLA4-Ig plus anti-LFA1 antibody infused
in vivo was immunosuppressive. However, although ourresults have shown that recipients of hCTLA4-Ig plus antiLFAl are not globally immunosuppressed as measured by
LDA analysis of anti-third party pPTL, these studies were
performed in the presence of exogenous IL-2 added to support the typically reduced
pPTL frequency observed postBMT. Therefore, based upon these data, we are unable to
assess whether the recipients that received hCTLA4-Ig plus
anti-LFAI in vitro and in vivo had antigen-specific hyporesponsiveness. The possibility that anti-LFA1 -directed approaches may lead to specific hyporesponses is suggested
by the experiments of Isobeetal”
who showedspecific
cardiac allograft acceptance with preservation of third-party
cardiac rejectioncapacityupon
challenge in micetreated
with antibodies to block the LFA1:ICAM pathways. Other
studies havesuggestedthattheblockade
of ICAM-I-induced signaling of LFAl on CD4’ T cells causes immunosuppressionratherthananergyinduction.I3
However, because these latter studies were performed with human CD4’
T cells andartificial APCs consistingof NIH-3T3 fibroblasts
transfected with a single MHC class I1 antigen (HLA-DR7),
it is unclear how extrapolatable these results are
to professional APCs that may have multiple non-B7 ligands capable
ofcostimulating T cells or to our
murine GVHD studies.
Finally, it isnoteworthythatthe
in vivo blockade of the
LFA1:ICAM signalingpathwaymight be advantageous in
BMT because other studies in mice have shown that antiLFAl
or
anti-ICAM
antibodies
can
facilitate
alloengraftment” and reduce murine lethal GVHD.” In human
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
CTW4 Ig and LFAI COBLOCKADE IN MURINE GVHD
BMT studies, anti-LFAl a chain antibody administered in
vivo to recipients of T-cell-depleted nonsibling donor grafts
has been shown to facilitate alloengraftment and survival in
children with immunodeficiency disorders.56The infusion of
anti-LFA1 a chain antibody to patients with steroid-resistant
severe acute GVHD was also found to be temporarily efficacious in 30% of patients5’
In summary, these data complement our recent studies in
which we have shown that the in vitro and/or in vivo modulation of the TCR on donor splenocytes by exposure to a
highly mitogenic anti-CD3c antibody is partially effective
in reducing lethal GVHD.58Both the previous5*and current
studies are examples of how the functional manipulation
of GVHD-causing donor T cells can reduce GVHD and
potentially avoid the complications of pan-T-cell depletion.
ACKNOWLEDGMENT
We thank Dr Marc Jenkins for valuable discussions and critical
reading of this manuscript.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 85: 2607-2618
Coblockade of the LFA1:ICAM and CD28/CTLA4:B7 pathways is a
highly effective means of preventing acute lethal graft-versus-host
disease induced by fully major histocompatibility complex-disparate
donor grafts
BR Blazar, PA Taylor, A Panoskaltsis-Mortari, GS Gray and DA Vallera
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