1. health and fitness
2. disease

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REVIEW ARTICLE
Pivotal Role of the B7:CD28 Pathway in Transplantation
Tolerance and Tumor Immunity
By Eva C. Guinan, John G. Gribben, Vicki A. Boussiotis, Gordon J. Freeman, and Lee M. Nadler
T
HE CLINICAL INTERFACE between immunology,
hematology, and oncology has long been appreciated.
Many conditions treated by the hematologist or oncologist
have either an autoimmune or immunodeficient component
to their pathophysiology that has led to the widespread adoption of immunosuppressive medications by hematologists,
whereas oncologists have sought immunologic adjuvants that
might enhance endogenous immunity to tumors. To date,
these interventions have generally consisted of nonspecific
modes of immunosuppression and immune stimulation. In
addition to the limited efficacy of these interventions, toxicities secondary to their nonspecificity have also limited their
overall success. Therefore, alternative strategies have been
sought.
Elucidation of the functional role of a rapidly increasing
number of cell surface molecules has contributed greatly to
the integration of immunology with clinical hematology and
oncology. Nearly 200 cell surface antigens have been identified on cells of the immune and hematopoietic systems.'
These antigens represent both lineage-restricted andmore
widely distributed molecules involved in a variety of processes, including cellular recognition, adhesion, induction
and maintenance of proliferation, cytokine secretion, effector
function, and even cell death. Recognition of the functional
attributes of these molecules has fostered novel attempts
to manipulate the immune response. Although molecules
involved in cellular adhesion and antigen-specific recognition have previously been evaluated as targets of therapeutic
immunologic intervention, recent attention has focused on
a subgroup of cell surface molecules termed costimulatory
molecules.*" Costimulatory molecules do not initiate but
rather enable the generation and amplification of antigenspecific T-cell responses and effector
In this review, we highlight one specific costimulatory pathway
termed B7:CD28.7"0 Since this ligandxeceptor pathway was
discovered 4 years ago, a large body of evidence has accumulated suggesting that B7:CD28 interactions represent one
of the critical junctures in determining immune reactivity
versus
In this review, we focus on preclinical
models that suggest ways in which this pathway might be
manipulated to induce either antigen-specific immunosuppression or immunostimulation.
COSTIMULATORY MOLECULES IN T-CELL ACTIVATION
AND FUNCTION
Requisite signals for T-cellactivation
and function.
Once T cells exit the thymus competent to respond to specific
antigen and enter the recirculating pool of cells in the peripheral blood and lymphoid tissues, they are capable of participation in both antigen-nonspecific and antigen-specific
events.'3-" Antigen-specific T-cell activation requires interaction of the T cell with specialized antigen-presenting cells
(~pc~).5,6,16-19
Depending on the microenvironment in which
the immune response is initiated, distinct populations of cells
Blood, Vol 84, No 10 (November 15), 1994: pp 3261-3282
serve as APCs. In the peripheral blood, for example, dendritic cells, activated B cells, and monocytes can present
antigen, whereas, in skin, keratinocytes and Langherans cells
serve this function. Because the major function of peripheral
blood dendritic cells, activated B cells, and activated macrophages is to process and present antigen, such cells are
termed professional A P C S . ' , ' ~ , ~ ~Other
, ~ ~ , *cells
' (eg, endothelium) can also present antigen under certain condition^.**^*^
Three distinct stages of cell-cell interaction between APCs
and antigen-specificT cells are required to induce an antigenspecific immune response. Figure 1 depicts the known cell
interaction molecules that are responsible for progression
from one stage to another. In a process termed adhesion,
APCs and T cells randomly interact both in the circulation
and in lymphoid tissues via cell surface ligands and their
receptor^.*^^*^ These ligands and receptors, referred to as
adhesion molecules, may be relatively lineage restricted (eg,
LFA-3 on APCs and its receptor CD2 on T cells) or, alternatively, they may be bidirectional (eg, ICAM-1 on APCs can
bind its receptor LFA-l on T cells and ICAM-1 on T cells
can bind LFA-1 on APC).*6.27Progression to the next stage,
termed recognition, occurs if the APCs can process, transport, and present sufficient quantity of the specific peptide
antigen in the context of the major histocompatibility complex (MHC).'8,29-33
Antigen-MHC will then be recognized
by the T cell via the T-cell receptor complex (TCR).30*33*34
Depending on the nature and source of the peptide antigen,
endogenous peptides (eg, derived from intracellular proteins)
are generally presented to T cells coupled to MHC class I
(HLA-A, B, or C), whereas exogenous processed peptide
antigens (eg, derived from circulating proteins) are generally
presented coupled to MHC class I1 (HLA-DR, DP, or DQ)
cell surface molecules.~8~*94~
Although there is a common
TCWCD3 complex, specific associative recognition structures on T cells are necessary to interact with the APC class
I or class I1 MHC. Antigens coupled to class I MHC molecules are recognized by TCWCD3 in the context of an associated CD8 molecule, whereas recognition of antigens coupled to class I1 requires CD4.4*,43This antigen-specific,
MHC-restricted interaction initiates a number of complex
signaling events and has been extensively reviewed by others.34.44-48
After ligation (ie, binding and cross-linking) of
TCR by antigen-MHC, T cells are competent to respond to
a number of potential second signals, termed costimula-
From the Division of Hematologic Malignancies and Pediatric
Oncology, Dana Farber Cancer Institute, and the Departments of
Medicine and Pediatrics, Harvard Medical School, Boston, MA.
Submitted June 23, 1994; accepted August 11, 1994.
Address reprint requests to Eva C. Guinan, MD, Department of
Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney St,
Boston, MA 021 15.
0 1994 by The American Society of Hematology.
0006-4971/94/8410-0036$3.00/0
3261
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3262
A-1
(CD
GUINAN ET At
-
LFA-1 ( C D l l d l 8 )
CAM-l (CD54)
ICAM-1 ( ~ ~ 5 4 )
VCAM
LFA-3 (CD58)
ICAM-1 (CD54 )
(CDll418)
CD43
VIA-4 (CD49d/29 )
HLA-AWC
Recognition
HLA-DR/DP/DQ
LFA-3 (CD58)
CAM-1 (CD54)
CD40
CD72
CD24
87-1 (CD80)
87-2 (CD86)
n[-]
Receptors
27
.;
D2
LFA-1 (CD11418)
CD40-L
CD5
CD28 CD24
CD28
CTLA-4
CTLA-4
tion.3-6.8.49.50Costimulatory molecules provide
T cells with additional signals that result in the initiation and enhancement of
pr~liferation.~,~'
Costimulation by some ligands results in cytokine production that can only be detected at the mRNA level,
whereas other costimulatory ligands are capable
ofinducing
significantsecretionandevenaccumulation
of ~ytokine?"'~
Figure 1 depicts a representative butnot exhaustive number of
candidate costimulatory molecules expressedon the surfaceof
@CS. Some costimulatory molecules appear to be expressed
on all professional Apcs, whereas others appear to be more
lineage restricted. Increasing evidence supports the notion that
several molecules previously considered to be adhesion molecules are also capable of delivering costimulatory signals (eg,
LFA-3, LFA-1, and 1CAh4-1)!~27~28~5742
The nature of costimnlatory signals and the potential therapeutic implications of their
modulation form the basis of this review.
Critical nature of the costimulatorysignal:two-signal
model of T-cell activation (Fig 2). Signaling through the
TCR is necessary but not sufficient to induce antigen-specific
T-cell activation and cytokine secretion.63@' In both murine
Fig 1. Antigen-specific interactions between APCs and T
cells are mediated byat least
three types of cell:cell interactions. A representative tiding of
receptors and ligands involved
in each of these steps is depicted.
and human systems, antigen-specific TCR signaling induces
a state of readiness in the T cell yet fails to induce proliferationand effector f ~ n c t i o n . ~ , 'This
, ~ ~ ,first
~ signal, termed
signal 1, is both antigen-specific and MHC-restricted. Signal
2, which is neither antigen-specific nor MHC-restricted, is
necessary to induce cytokine secretion, cellular proliferation,
and effector f u n ~ t i o n . ~The
, ~ .critical
~ ~ nature of this second
costimulatory signal was originally proposed by Bretscher
andCohn?' In their two-signal model, later extended by
Jenkins and S c h ~ a r t z , 6 ~both
- ~ ' a TCR and a costimulatory
signal are essential for T-cell clonal expansion, lymphokine
secretion, and effector function. If signal 2 is not delivered,
T cells enter a state of long-term unresponsiveness to specific
antigen-termed
One hallmark of this state is
the inability of anergized T cells to secrete interleukin-2 (IL2), although they retain their capacity to proliferate in the
The molecular nature
presence of exogenous IL-2.2*6."3,62.66
of the second signal(s) necessary to induce cytokine secretion and cellular proliferation and thus prevent anergy eluded
immunologists for nearly two decade^.',^'
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3263
PIVOTAL ROLE OF B7
Immune Response
Receptor
IL-2
A
1
Fig 2. Theupperhalfof
the
of
figuredepictspresentation
antigen by a professional
APC to
a T cell. Because signal
1 and rignal2 ara delivered,11-2 secretion
and upregulation of and signalIL-2R occur.
The
ingthrough
lower half of the figure depicts
presentation of the same antigen by an APC lacking costimulatory IigandM. Because
only
signal 1 is delivered, significant
11-2 accumulation and upregulation and signaling throughIL-2R
fail to occur, multing in the generation of antigm-specific anergy.
Evidence that the B7 molecule expressed on APCs delivers
signal 2 through the T-cell sulface molecule CD28. After
delivery of signal 1, ligation of the CD28 molecule expressed
on T cells results in the delivery of signal 2 and progression
to T-cell proliferation, cytokine secretion, and effector funct i ~ n .CD28
~ . ~ is a homodimeric glycoprotein member of the
Ig supergene family of cell surface interaction molecule^^.^.^^
and has a single IgV domain (Fig 3).67 The expression of
this molecule is unusual as it is limited to T lymphocytes
and plasma
CD28 is expressed on virtually all thymocytes that coexpress both CD4 and CD8 molecules and
is also expressed on 80% of mature peripheral blood T
~ e l l s . ~In~ the
, ~ *human, nearly 95% of CD4+ but only 50%
of CD8+ peripheral blood T cells are CD28+.73Both antigenspecific signals via the TCR and nonspecific signals delivered by mitogens significantly upregulate the expression of
CD28.8.68,74-76In humans, a very small subset of CD4+ and
50% of CD8+ T cells are CD28-. Most of these CD28- T
cells express an antigen, CDllb, often found on myeloid
~ e l l s . The
~ ~ ,function
~~
of these cells is presently unknown,
although some reports suggest that they have noncytotoxic,
suppressor
For nearly a decade, it has been known that the CD28
molecule is involved in the induction of T-cell proliferation
and IL-2 secretion. Although cross-linking CD28 on resting
T cells with anti-CD28 monoclonal antibody (MoAb) induced no apparent T-cell activation or change in function,
cross-linking CD28 after either TCR signaling or nonspecific
activation of T cells with mitogen resulted in markedly enhanced T-cell proliferation and cytokine secretion.6837"76*81-83
Cross-linking of the CD28 molecule also provided a costimulatory signal for T cells activated via the CD2 pathway.54*84-87
Neither the signal transduction pathway nor the
transcriptional pathway are fully ~nderstood.~~~.~~.~~~'~'
However, candidate mechanisms of CD28-mediated signaling
have been recently reviewed and suggest an important role
for phosphatidylinositol 3-kina~e.~."~
Functionally, CD28
signaling has been found to increase secretion of the
cytokines IL-la, L-2, interferon-y, IL-3,granulocyte-macrophage colony-stimulating factor (GM-CSF), and lymphotoxin.3.8.51-53.91.95,104.105
This results from at least twoCD28mediated effects most thoroughly studied in the case of
IL-2 in which prolongation of E - 2 mRNA half-life and
upregulation of IL-2 transcription result in accumulation of
considerable amounts of secreted IL-2.5L.74,90*9'.95.'"
Indeed,
T cells isolated from mice deficient in the CD28 molecule
(so called CD28 knock-out mice) have decreased response
to lectin mitogens and absent L - 2 secretion in response to
lectin acti~ation.'~'
In addition, they have abnormal T-cel1:Bcell interactions as shown by decreased Ig production and
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3264
GUINAN ET AL
CD28
COOH COOH
COOH
Gene Location
2q33 - 2q34
87-2
I
I
COOH
CTLA-4
l
NH2
NH2
COOH
Gene Location
2q33
87-1
Gene Location
2q33
B7-X
3
COOH
Gene Location
3q13.3- 3q21
COOH
Gene Location
3ql3.3 - 3q21
Fig 3. The top panel shows a
schematic ofthe predictedstructure CD28
of
and CTLAJ.
Whereas CD28 is homodimeric,
CTLA-4isdepicted in both homodimeric and monomeric configurations. The bottom panel
shows the predicted structures
of members of the B7 family.
Several linesofevidencesuggest that additional CD28KTLA4 counter-receptors exist (B7-x).
However, they are omitted becausethesegeneshaveyettobe
cloned.
abnormal Ig regulation. Despite these defects, the mice appear interaction resulting in costimulation (signal 2).ll3The prototo have relatively normal cellular immunity, including cytotoxic type CD28 counter-receptor expressed on APC, B7,was
T-cell and delayed-type hypersensitivity responses, suggesting
initially discovered on activated but not resting B cells and
the existenceof redundant pathways providing important costiwas thought to be restricted to activated B cells."4."5 Molecular cloning of the original B7 molecule (now termed B7mulatory signals. Further exploration
of this model system may
yield a greater understanding of the role CD28 plays in both
1, CD80) showed that its gene product was a member of the
normal development and immune challenge because the func- Ig supergene family with two Ig-like domains (IgV and IgC,
Fig 3).116.117 The B7-l gene was subsequently localized to
tional consequences of CD28-mediated signaling and subsechromosomal region 3q13.3-3q21.II8 Further investigation
quent IL-2 secretion are likely to be quite complex.
showed that the B7 molecule was not, in fact, B-cell reHowever, even without full delineation of these consestricted as it could be induced on monocytes by interferonquences, observations that the CD28 signaling pathway is
not inhibited by cyclosporin suggested that reagents capable
y.'I9 B7 has also been found on some human T-cell clones
of modulating CD28 signaling might be of relevance in preand repetitively activated human T lymphocytes in which it
vention of tissue rejection and graft-versus-host disease
can be specifically induced by either IL-7 or anti-CD3 MoAb
(GVHD).52.'05.'08I12 However, development of such potential
cross-linking of the CD3/TCR c ~ m p l e x . ' ~ ~Considerable
"~'
additional confusion concerning the lineage restriction of the
therapeutics awaited the discovery of the CD28 counterB7 molecule arose from the observation that two different
receptor on APCs. Subsequent manipulation of this receptor:ligand pair permitted unequivocal demonstration that
MoAbs [anti-B7 (133) and BB11 both reacted with the gene
product of the B7gene."' Whereas anti-B7 (133) MoAb
CD28 represented the T-cell component of the T-cel1:APC
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PIVOTAL ROLE OF B7
3265
human systems suggested that more than one B7 molecule
existed. First, anti-B7 (133) MoAb was not as effective as
CTLA4-Ig in inhibiting costimulation provided by professional APCs.I5' Second, different MoAbs, thought to identify
the same B7 antigen (as discussed above), did not demonstrate equivalent staining of target cells or tissues. For example, keratinocytes could be stained with BB1 but not with
anti-B7 (133) MoAb.'2"'26 Further study showed that these
BBl-positive keratinocytes didnot express B7-1mRNA,
strongly suggesting that these MoAbs defined distinct molecules.Iz6 Finally, APCs isolated from B7-l-deficient (B7
knockout) mice remained capable of both providing a costimulatory signal, albeit somewhat reduced, and binding
CD28 MoAbs or cells expressing B7 induced T-cell proliferCTLA4-Ig.153All these lines of evidence argued strongly for
ation and cytokine secretion. Moreover, anti-CD28 Fab or
additional members of the B7 family. Several investigators
anti-B7 MoAb inhibited proliferation and IL-2 secretion inthen showed that murine andhuman
APCs expressed
duced by APCs or allogeneic cells in mixed leukocyte reactions.56.132,138.13YInterestingly, there is enormous phylogenetic
CTLA4-Ig binding ligands that were not the original B7
m o l e c ~ l e .Within
~ ~ ~ -several
~ ~ ~ months, two laboratories had
conservation of the binding domains of the molecules in this
cloned an identical alternative B7.'4'*158*159
Freeman et a1159.'60
p a t h ~ a y . " ~For
* ' ~example, mouse B7binds and signals
termed the original B7 molecule B7-1 andthe new B7through CD28 on human T cells and human B7 both binds
like molecule B7-2 (CD86). The B7-2 gene has also been
to and signals through mouse CD28.1'7.'40.'4'
localized to chromosomal region 3q13.3-3q21.161.162 Despite
Complexity of the B7:CD28 familiesof cell surface molesharing the identical Ig supergene family structure with an
cules. As the importance of this pathway to T-cell activaIg variable (IgV) and Ig constant (IgC) region (Fig 3) and
tion was investigated, it readily became apparent that additional molecules were involved. On the T-cell side, Brunet
sharing the ability to bind CD28 and CTLA-4, these molecules were not highly homologous at the DNA Level."'
et all4'cloned a gene encoding a molecule highly homoloIncreasing evidence in murine and human systems suggous to CD28 that they termed CTLA-4. Sequence analysis
gests that B7-1 and B7-2 may have distinct costimulatory
predicted that the protein product would be a glycoprotein
functions,158.159,163This hypothesis is supported by observamember of the Ig supergene family that, like CD28, would
tions detailing differences in the temporal expression of these
have a single IgV-like domain (Fig 3). Recent evidence sugmolecules after B-cell activation. In both mouseand human,
gests that it may exist as a monomer or homodimer on the
cell surface, but little, if any, evidence suggests that it can
neither B7-1 nor B7-2 protein is expressed on unstimulated
B cells, although low levels of B7-2, but not B7-l, mRNA
form a heterodimer with CD28.'43CTLA-4 and CD28 appear
to be a gene duplication because both were found very close
are p r e ~ e n t . ' ~B7-2
~ . ' ~appears
~
on the cell surface within 24
together on chromosome 2.'44-147
Linsley et all4*showed that
hours of B-cell activation and B7-1 appears later.'4'."7.163
CTLA-4, like CD28, bound to B7 and that a soluble fusion
Further discordance has been observed in unstimulated huprotein composed of the extracellular domain of CTLA-4
man monocytes in which B7-2 is constitutively expressed
and the human Ig Cy1 chain, termed CTLACIg, blocked
whereas B7-1 expression is induced after activation.'"
B7-mediated costimulation. Surprisingly, B7 had a much
Finally, we and others believe that the story is still not
higher affinity for CTLA-4 than for CD28, leading some
complete. We have reported thatBB1 MoAb appears to
investigators to refer to CTLA-4 as the high-affinity receptor
recognize a third CTLA-4 binding molecule, which is now
for B7.I4'In contradistinction to CD28, neither CTLA-4
termed B7-3.157This molecule has not yet been molecularly
mRNA nor cell surface protein could be detected in resting
cloned and, therefore, its structure and function cannot be
T cells, although both appeared after a c t i v a t i ~ n .CTLA~ ~ ~ ~ ' ~ ~addressed. Additional members of the B7 and CD28/CTLA4, therefore, appears to be coexpressed with CD28 on the
4 families may also exist. Because CTLA-4 does not appear
surface of activated T cells. The functional role of CTLA-4
to provide the redundant costimulatory signal in the CD28has been difficult to clarify. The absence of IL-2 production
deficientmouse, investigators are searching for either anin the CD28 knock-out mouse suggests that CTLA-4 neither
other counter-receptor for B7 family members or even anfunctions equivalently to CD28 nor does it provide a CD28other distinct costimulatory pathway.Io7
like signal 2 equivalent. These findings are consistent with
Model of B7:CD28-mediated costimulation and levels of
the hypothesis that CTLA-4 does not appear to be a CD28potential immune intervention. Before addressing the poredundant pathway.lo7 Several investigators have reported
tential pathophysiologic relevance of this pathway, we will
that, although cross-linking human CTLA-4 does not provide
present a hypothetical model (Fig 4) of the molecular interaca primary costimulatory signal, it does enhance the costimutions that occur at distinct stages of APC:T cell interaction,
latory signal delivered byCD28.I5'
Others suggest that
including (I) adhesion and antigen presentation, (11) TCR
CTLA-4 might provide a negative signal.'" Therefore, the
signaling, (111) costimulation, and (IV) cellular proliferation
relationship between CD28 and CTLA-4 in the regulation
and function.
of T-cell activation is still under active investigation.
After random cellular adhesion mediated by adhesion liOn the APC side, several lines of evidence in murine and
gands and receptors. APCs that have antigen associated with
appeared to stain only professional APCs, BB1 MoAb also
stained thymic epithelium, keratinocytes, and Langerhans
These data provided support for the thesis that,
in the human, anti-B7 (133) and BB1 MoAbs might define
distinct, but antigenically cross-reactive, molecules.
Linsley et
found that cells transfected with CD28
could adhere to cells transfected with the B7 molecule and
that binding could be blocked by either anti-CD28 or BB1
MoAbs, definitively showing that these molecules were a
receptorligand pair. Evidence that B7 provided signal 2 via
CD28 then accumulated rapidly in both human and murine
systems,82.117.130-137
Here, ligation of CD28 by either anti-
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3266
GUINAN ET AL
APC
T Cell
I. Adhesion
and antigen
presentation
IL-2R mRNA
11. T cell
receptor
signaling
IL-2 mRNA
111. Costimulation
IV. Cellular
proliferation
and function
their MHC are poised to trigger antigen-specific T cells via
the TCR (stage I). Engagement of the TCR by antigen initiates a series of events (signal 1) that upregulates CD28 expression and induces IL-2 receptor (IL-2R) expression and
IL-2 mRNA transcription (stage 11). Cells receiving signal
1 are capable of low levels of proliferation and, therefore,
appear to secrete IL-2 in an autocrine but not a paracrine
fashion. On the APC side, the signal(s) that induces human
B7-2 expression are still under investigation. However, once
CD28 is upregulated and B7-2 is expressed, costimulatory
signaling through the CD28 molecule occurs (signal 2, stage
111). Within 24 hours of signal 2, the T cell begins to secrete
cytokines (perhaps most importantly IL-2) in a paracrine
fashion and to express surface CTLA-4. Over the next 24
hours, T-cell proliferation is markedly enhanced and B7-1
Fig 4. A hypothetical model
of the various stagesof antigenspecific activation of T cells by
APCs. In stage 1, adhesion receptodigand interactionsjoin APCs
and T cells. Antigen (Ag) is digested,
processed,
and
presented by APCs in the context of
MHC. In stage II, the APC delivers
signal lto the T cell through the
TCR complex initiatingtranscription and expression of
low levels
of IL-2 and IL-2R.
During stage 111,
67-2CD28-mediated costimulation delivers signal 2, thus s i g
nificantly upregulating 11-2 production and IL-2Rexpression.
Stage IV alludes to further
APCT-cell contact-generatedinteractions, most of which are
notwell
understood, which
further amplify or potentially
downregulatethe T-cell proliferative and effector response. Additional CD28lCTLA-4 binding
molecules lindicated as 67-x)
may participate in these interactions.
and other CD281CTLA-4 counter-receptors (B7-x) appear
on the APC surface. The functional consequences of CTLA4, B7-1, and B7-x expression and their potential interactions
are still to be elucidated. Regardless of the precise contribution(s) of each of these molecules, costimulated T cells proliferate and are functionally competent. Which precise signals direct the T cell toward amplification ofan immune
response or drive the T cells toward memory are poorly
understood.
Albeit incomplete, Fig 5 depicts the known interactions
between an APC and an antigen-specific T cell and proposes
several stages at which intervention might result in inhibition
of the immune response. Specifically, it is possible to block
either adhesion, TCR signaling, or B7 family-mediated costimulation through CD28. Although each maneuver results
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3267
PIVOTAL ROLE OF B7
~~
Outcome
Site of Intervention
APC
T cell
Antigen
Recognition
Proliferation
Proliferation
on rechallenge
+
Potential
therapeutic
reagents
alCAM-1
a LFA- 1
a LFA-3
CD2-Ig
8ioc&Adhesion
-
+
aTCR
aHLA-DR/DP/DQ
a HLA-A/B/C
Bfock Signal 7
Bfock Signa/ 2
aB7-1
a 87-2
&D28 Fab
CTLA4-Ig
CD28-lg
Soluble CTIA-4
Fig 5. Three sites of intervention in APC:T-cell interaction are depicted along with available reagents capable of blocking at each site.
Reagents blocking either adhesion orTCR signaling (signal 1) prevent antigen recognition and T cells remain naive. In contrast, reagents
blocking signal 2 do not affect antigen recognition but do prevent both the primary proliferative response and subsequent antigen-specific
proliferation resulting in the induction of anergy.
in inhibition of T-cell proliferation, the resulting capacity of
T cells to respond to antigen on rechallenge differs significantly (Fig 5). Inhibition of one or more critical adhesion
interactions, either by blocking the adhesion ligand or its
receptor (stage I in Fig 4), completely abrogates the ability of
T cells to receive a signal via their antigen or costimulatory
receptor and, therefore, the immune response is totally inhibited. In fact, blockade of adhesion completely prevents the
recognition of antigen. Moreover, once the adhesion blockade is removed, these T cells respond on rechallenge asif
they had never before encountered the antigen, ie, they behave like naive T cells mounting a primary immune response. Similarly, if adhesion is intact but TCR signaling
is prevented (stage I1of Fig 4), no antigen recognition or
proliferation occurs and removal of TCR signaling blockade
results in a primary response after rechallenge with antigen.
Thus, the functional outcome of blockade of adhesion or
TCR recognition is immunosuppression. If these T cells are
withdrawn from such inhibitory conditions, they are again
capable of responding to the initial specific antigen. However, if the blockade is at the level of the B7:CD28 costimulatory pathway, then the outcome is quite different. After
effective adhesion and TCR signaling, T cells must receive
a B7 family-mediated costimulatory signal (signal 2) to pro-
liferate and become functional (stages I11 and IV of Fig 4).
Blockade of CD28 or B7 family members is sufficient to
inhibit signal 2, resulting in failure of both proliferation and
IL-2 secretion (Fig 5 ) . T cells that do not receive a costimulatory signal via CD28 enter a state of long-term antigenspecific unresponsiveness and are unable to respond to
rechallenge with the antigen. In summary, blockade of adhesion or TCR signaling results in immunosuppression,
whereas blockade of the B7:CD28 pathway results in the
induction of anergy.
Because it might be advantageous to suppress or even
anergize an immune response in a number of clinical settings, a panel of potentially clinically effective reagents have
been developed that might inhibit to a greater or lesser extent
the receptor:ligand interactions contributing to the above
stages of T-cell activation. To date, the most effective agents
include MoAbs and fusion proteins. As the signal transduction pathways become better elucidated, small molecules,
analogous to cyclosporin, will certainly figure inthe modulation of these events. Figure 5 summarizes the available specific reagents and the stages of APC:T cell interaction that
they are likely to interrupt. Extensive evaluation of in vitro
and several murine preclinical models has been undertaken
with each of these reagents.
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3268
INDUCTION OF TRANSPLANTATION TOLERANCE BY
BLOCKADE OF THE B7:CD28 COSTIMULATORY
PATHWAY
Inan effort to decrease the morbidity and improve the
results of transplantation of both solid organs and bone marrow, investigators have attempted to develop methods to
induce a state of long-term antigen-specific unresponsiveness. This state has beentermedanergyin
vitro and
tolerance in v i ~ o . ~ ~ , ~Specifically,
,~""~
anergy reflects the
inability of antigen-specific T cells stimulated through their
antigen receptor to mount a secondary antigen-specific response on rechallenge. Tolerance is the identical response
reflected in the inability of the intact host to mount an effective secondary antigen-specific response in vivo. Historically, experimental methods to induce anergy involved fixation or chemical modification of APCs in vitro to destroy the
ability of APCs to provide signal 2.168.16y
These techniques,
although effective, were not readily translatable to the clinical arena. On the other hand, clinically available agents such
as cyclosporin A and FK506 have been shown to be ineffective in blocking signal 2.1fls*109~11fl~'7fl
Therefore, such reagents
are capable only of inducing immune suppression but not
long-lived antigen-specific tolerance. In this section, we will
review preclinical murine and human studies suggesting that
blockade of the B7:CD28 costimulatory pathway is sufficient
to induce anergy in vitro and tolerance in vivo and propose
potential therapeutic reagents and settings that might be appropriate for clinical experimentation.
Induction of anergy in vitro. Compelling evidence
shows that blockade of the B7:CD28 pathway is involved
in the generation of anergy in both human and murine in
vitro systems.S6.132.139.152.166
In several experimental settings,
artificial APCs (nonhematopoietic cells transfected with
MHC and B7 molecules) were able to function effectively
as APCs and induce antigen-specific proliferative responses
accompanied by IL-2 accumulation in both human T-cell
clones and normal human T l y m p h o ~ y t e s . ~ ~ Incubation
,"~.'~~
of these same T cells with the identical specific antigen in
the context of an artificial APCs bearing MHC but not B7
was undertaken to model delivery of signal 1 but not signal
2. T cells stimulated in this way did not secrete appreciable
amounts of IL-2 and were induced into a state of anergy.","'
Anergy could also be induced by blocking the capacity of
B7 family members to signal through CD28.56,132,139
This
blockade could be effected using inhibitory reagents including (1) combinations of individual blocking MoAbs to B7
family members; (2) CTLA4-Ig, the soluble high-affinity
counter-receptor for B7 family members that also interrupts
B7 binding to CD28; and (3) anti-CD28 Fab, which similarly
prevents B7 binding to CD28. Importantly, anergized cells
were capable both of proliferation and secretion of IL-2 after
stimulation with mitogen and of proliferation in response to
exogenous IL-2, again showing that the anergized cells were
functionally intact and that their unresponsiveness was limited to specific antigen." Most importantly, the reciprocal
experimental conditions, ie, simultaneous engagement of the
TCR by antigen and either ligation of CD28 with MoAb or
exposure to cells expressing the B7-1 molecule, averted the
GUINAN ET AL
induction of anergy.56.'32,'3y
These studies provided the first
evidence that CD28 costimulation was a signal critical to
the induction of a full immune response and, importantly,
that its absence during antigen-specific TCR signaling resulted in induction of the anergic state. In addition, these
experiments showed that blockade of this pathway was able
to induce anergy in previously sensitized T cells, ie, T-cell
clones. These observations were surprising yet supportive of
the notion that CD28-mediated costimulation isnot only
essential for primary T-cell activation but also necessary for
antigen-specific T-cell reactivation.
Subsequent studies showed that B7 family-mediated costimulation was,in fact, uniquein its ability to prevent T
cells from entering the anergic state. To determine the contributions of individual costimulatory molecules toprimary
and secondary immune responses, humanmodel systems
using transfection of specific alloantigens and costimulatory
molecules were developed to study the presentation of isolated alloantigen in the presence of either B7- 1 or ICAM-1 .'('
Although alloantigen in the presence of either costimulatory
molecule, B7-1or ICAM-I , induced equivalent levels of
proliferation, several lines of evidence suggested that these
costimulatory signaling pathways differed. First, B7-1 -mediated costimulation was cyclosporin resistant, whereas
ICAM- 1-induced proliferation was abrogated by cyclosporin. Second, B7- 1 costimulation was accompanied by accumulation of IL-2in the culture supernatants, whereas
IL-2 accumulation could not be detected after ICAM-I costimulation. Finally, and most importantly, subsequent antigen-specific rechallenge of the T cells resulted in a classic
secondary response only when B7-I costimulation had been
provided during primary stimulation. In contrast, when
ICAM- 1 had delivered the initial proliferative costimulatory
signal, T cells were anergic on rechallenge. The addition of
third-party alloantigens, exogenous IL-2, or mitogens to the
secondary culture resulted in proliferation, showing that the
anergized cells were viableand capable of responding to
several stimuli but not to specific antigen. Of note, the addition of exogenous IL-2 to primary cultures under conditions
normally producing T-cell anergy resulted in development
of an antigen-specific T-cell proliferative response on rechallenge. This finding suggested that IL-2 could functionally
replace CD28-mediated costimulation, supporting the contention that CD28-mediated regulation of IL-2 secretion and
perhaps IL-2R expression are pivotal in the prevention of
the anergic state. These CD28-mediated IL-2-related effects
may explain why blockade of other costimulatory and/or
adhesion molecules appears to limit primary immune responses but only effective blockade or absence of B7:CD28
interaction induces antigen-specific anergy.
Additional evidence that blockade of the B7:CD28 pathway during a primary alloantigen-specificsensitization limits
responsiveness on rechallenge comes from the experiments
of Tan et al,Is2 who studied human mixed-leukocyte responses (MLR) between fully HLA-disparate individuals.
The presence of either anti-CD28 Fab or CTLA4-Ig in the
primary MLR significantly inhibited response to specific alloantigen on rechallenge whereas responsiveness to third
party cells was not modified. Unlike results observed above
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PIVOTAL ROLE OF B7
using alloreactive T-cell clones, interruption of the B7:CD28
pathway did not result in anergy, although a 50% to 85%
inhibition of secondary proliferation was observed. Generation of cytolytic T-cell precursors was also inhibited. Of
note, anti-B7-l MoAb was less effective (30% inhibition of
proliferation). In this system, IL-2 (as assayed by presence
of IL-2 mRNA) was completely suppressed, although IL-4
mRNA was detectable. As in the above experiments, exogenous IL-2 induced proliferation of the hyporesponsive T
cells. Moreover, the addition of IL-2 to the primary sensitization prevented alloantigen hyporesponsiveness on rechallenge. Gribben et all" have confirmed the above observations. Furthermore, they have demonstrated that the addition
of CTLACIg results in substantial decreases in the frequency
of precursor helper T cells generated during MLR between
both matched and mismatched family donor:recipient pairs.
These and the above results provide a rationale for attempting to inhibit the B7:CD28 pathway in vitro or in vivo
to prevent allograft rejection or GVHD.
Induction of tolerance. In fact, several in vivo organ
transplant models support the hypothesis that blockade of
the B7:CD28 pathway will induce a long-term state of donorspecific tolerance. In one model of xenogeneic pancreatic
islet cell transplantation, mice were treated with streptozotocin to eradicate their pancreatic islet /?-cell f~nction.'~'
Human pancreatic cells were then transplanted under the
kidney capsule. If the transplant was successful, islet cells
would produce sufficient insulin to reverse hyperglycemia.
In an attempt to prolong the survival or prevent the rejection
of human pancreatic islet cells, mice were treated with either
intravenous human CTLACIg (which binds to both human
and mouse B7) or control Ig fusion protein from the time of
transplant through the ensuing 14 days. CTLACIg treatment
resulted in normal functioning of the transplanted islet cells,
and biopsies of the islet xenografts showed intact islets without lymphocytic infiltration. In contrast, control animals had
a mean graft survival of only 6 days and islet tissue showed
significant lymphocytic infiltration and islet destruction. Donor-specific tolerance was demonstrated by nephrectomy of
the graft-bearing kidney and transplantation (without
CTLA4-Ig) of pancreatic islet cells from either the original
xenograft donor or an unrelated donor under the remaining
kidney capsule. Xenogeneic islets from the original donor
survived and functioned, whereas islets from the unrelated
donor were rejected by day 5. Thus, CTLACIg treatment,
without the need for any additional immune manipulation,
resulted in long-term, donor-specific tolerance to xenoantigen and formally demonstrated that tolerance could result
from in vivo blockade of the B7:CD28 pathway.
In a second model, CTLA4-Ig was used in an attempt to
induce tolerance in a fully mismatched rat cardiac allograft
Preliminary experiments showed nearly complete suppression of a primary MLR by human CTLACIg.
In contrast to the pancreatic islet cell xenografts, the cardiac
allografts were ultimately rejected, although infusion of
CTLACIg resulted in prolongation of graft survival.'74The
failure of CTLA4-Ig to induce tolerance to alloantigen could
be interpreted in a number of ways, including (1) CTLA4Ig was immunosuppressive butdidnot induce anergy, (2)
3269
anergy was induced and then reversed, or (3) the model
system did not permit effective tolerization of resident host
T cells because of inadequate presentation of alloantigen,
particularly because cardiac tissue isunlikelyto function
well as an APC. This last hypothesis was tested by modification of the experimental design to include donor splenocyte
infusions at the time of cardiac transplantation followed by
CTLACIg treatment 2 days later.'73This design represented
an attempt to assure that all, or nearly all, host T cells had an
opportunity to efficiently "see" donor alloantigen (receive
signal 1) by increasing the number of available APCs expressing relevant donor alloantigens. Delivery of signal 2
would then be blocked by infusion of CTLACIg. This maneuver resulted in long-term viability of the cardiac allograft,
whereas treatment with either CTLA4-Ig alone or donor
splenocytes alone produced graft prolongation but not longterm graft survival. Only mononuclear cells syngeneic to the
cardiac allograft were effective in preventing rejection; thirdparty mononuclear cells provided no protective effect. These
studies provide compelling evidence that, under optimal circumstances, blockade of the B7:CD28 pathway can lead
to antigen-specific tolerance facilitating long-term allograft
survival without the need for additional immunosuppression.
Early results of experiments investigating the effect of
CTLACIg in a murine bonemarrow transplant (BMT)
model suggest that blockade of this pathway will have some
prophylactic efficacy with respect to GVHD.'75BMT of donors and recipients completely mismatched at both class I
and class I1 MHC was undertaken with or without in vivo
administration of CTLACIg. Hematopoietic reconstitution
was unaffected and CTLACIg treatment consistently reduced the incidence of lethal GVHD in recipients, although
most animals had evidence of subclinical disease. These
studies suggest that CTLACIg can be well-tolerated in the
BMT setting and that its use should be further explored to
establish the most efficacious treatment regimen. Furthermore, these observations should be extended to models with
less MHC disparity, thus better approximating siblingmatched and family-mismatched BMT.
Reversal of anergy. The potential therapeutic benefits of
inducing alloantigen specific tolerance in the settings of BM
and solid organ transplantation are dependent on the longterm stability of the anergic state. In vitro studies show that
anergy to specific antigen can be reversed in murine T-cell
clones when anergized clones are cultured for prolonged
periods in IL-2 and then exposed tothe original specific
antigen presented by professional APCs.17' In humans, similar data in an alloantigen-specific clonal T-cell model bolsters the observation that prolonged exposure to IL-2 may
initiate or facilitate reversal of anergy.'77In fact, organ rejection and either onset or recurrence of GVHD are frequently
preceded by infections that undoubtedly induce nonspecific
inflammation and cytokine re1ea~e.l~'Thus, nonspecific
events such as intercurrent infections and, potentially, antigen-specific events such as activation of IL-2 secretion
through alternative pathways may both pose formidable obstacles to maintenance of the anergic state. However, even
if it proved impossible to maintain the anergic state, it is to
be hoped that induction of even a limited period of tolerance
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3270
would allow naive T cells produced during the quiescent
state to be "educated" to see either the allogeneic graft or
host as ~ e 1 f . I ~ ~
POTENTIAL ROLEFORB7
FAMILYMEMBERSINTHE
INDUCTION OF TUMOR-SPECIFIC IMMUNITY
Potential capacity of human tumors to present antigen.
Although immunologists have identified, and in certain circumstances expanded, small numbers of antigen-specific T
cells capable of inducing cytolysis of human neoplastic cells,
there is little evidence that these cells are important for autologous tumor surveillance or tumor r e j e c t i ~ n . ' ~It~is" ~un~
clear why we fail to mount an antigen-specific T-cell-mediated immune response against malignant cells and, if one is
mounted, why it fails. Of course, the hypothesis that a host
can generate a significant cytotoxic T-cell response directed
against its autologous neoplasm presumes the existence of
sufficient numbers of antigen-specific helper and cytotoxic
T-cell precursor^.'^'.'^^ For the purposes of this review, we
will presuppose sufficient numbers of T-cell precursors and,
therefore, we will concentrate our discussion on the multiple
theoretical reasons why neoplasms may not effectively elicit
T-cell-mediated tumor-specific immunity. In keeping with
this focus, we will discuss the generation of effective antitumor immunity from the vantage point of the potential capacity of the tumor cell to present antigen and emphasize experiments examining the role of costimulation in the generation
of tumor-specific tolerance or cytotoxicity (Fig 6).
In the specific case of leukemias and lymphomas, malignant cells are often the neoplastic counterparts of professional APCs, suggesting that these cells ought to have the
repertoire necessary to effectively present endogenous tumor
antigens to T cells.l14.116.180.187-193However, the failure of any
given tumor to elicit an adequate, tumor-specific T-cell response might result from (1) absent or inadequate expression
of adhesion molecules; (2) inability to effectively present
antigen consequent to absence of a tumor antigen, lack of
immunogenicity of the antigen, andor inadequate processing
or transport of antigen; (3) absent or limiting cell surface
MHC; and (4) absent or limiting cell surface costimulatory
molecules, specifically B7. These defects would result in
discrete and different sequelae and might be amenable to a
variety of therapeutic manipulations, depending on the site
of the d e f e ~ t . ' ~ ~ " Potential
~ ~ . ' ~ ~defects
. ' ~ ~ in antigen presentation by neoplastic cells are depicted in Fig 6A. If tumor
cells lack sufficient numbers of adhesion receptors, MHC
molecules, or antigen, then no signal 1 is generated. Therefore, no T-cell-mediated immune response is induced. In
contrast, if both adhesion and signal 1 delivery mechanisms
are intact, absence of signal 2 results in tumor-specific tolerance. In this section, wewill summarize both theoretical
constraints on the ability of neoplastic cells to elicit adequate
T-cell-mediated antitumor activity and in vitro and in vivo
murine experiments that begin to elucidate the conditions
under which either naive or tumor-bearing hosts can be induced to recognize and kill syngeneic tumor.
Little is known about the expression of adhesion ligands
and receptors by solid tumor cells. In contrast, several hematologic malignancies have been extensively studied for their
GUINAN ET AL
expression of adhesion molec~les.'~l
Different histologic
subtypes of non-Hodgkin's lymphomas (NHLs) appear to
have relatively characteristic and distinct profiles of adhesion
receptor surface expression. Not infrequently, neoplastic B
cells lack one or more of the adhesion receptors commonly
expressed on either normal pre-B or mature B cells; the
extreme example is Burkitt's lymphoma, which has no or
limited expression of L-selectin, CD44, LFA-I, ICAM- 1, or
LFA-3.I9' Of the other components necessary to facilitate
antigen presentation, the expression of class I and class I1
antigens by tumor cells has been most systematically examined. 195.196 A significant proportion of epithelial tumors show
complete or partial loss of class I expression in comparison
to the tissues from which they a r o ~ e . ' ~This
~ . ' is
~ ~less well
studied in hematologic malignancies, although approximately half of B-cell NHLs appear to lack cell surface class
*.192,193.19s Class I1 expression, in contrast, may be more frequent in epithelial tumors than in their normal counterparts.'" Hematologic malignancies vary greatly in their
expression of class I1 (as ascertained by immunophenotyping)~1Y0.192,193The vast majority of B-cell malignancies are
class I1 positive, as are normal B cells, whereas the majority
of T-cell malignancies are
Expression of class I1
in acute myeloid malignancies is highly variable (eg, more
common in French-American-British [FAB] M 1 and M2 and
uncommon in M3 and M4) and poorly predictable.'" Like
their normal cellular counterparts, neoplastic cells can upregulate class I and class I1 after exposure to interferons.'"-""
The most controversial aspect of the potential ability of
malignant cells to generate antigen-specific immunity is
the very existence and detection of tumor-specific antigenS.183-IK6.2M).201
Tumor antigens have been functionally identified in experimental systems in which naive mice are inoculated (immunized) with irradiated killed
ortumor
ce~~s.183,18S,186,194.202
The same mice are subsequently inoculated (rechallenged) with unmanipulated tumor cells. Tumor
growth is then assessed. Tumors that are rejected or are
limited in their growth in comparison to their growth in
unimmunized control animals are defined as immunogenic,
whereas those that growequivalently in immunized and nonimmunized hosts are defined as nonimmunogenic. In these
models, generation of appreciable antitumor activity against
so-called immunogenic tumors generally requires repetitive
courses of immunization. This suggests that, although the
tumor cell itself may be incapable of functioning efficiently
as an APC, shedtumor antigens, if present in sufficient
quantity, may be taken up, processed, and presented by normal host professional APCs with subsequent generation of
an effective, tumor antigen-specific immune response. The
tumor antigens themselves have generally not been identified
in these experiments. Until recently, the existence of human solid tumor-specific antigens wasnotwell substantiated,18%186The paradigm of tumor-specific antigens in humans evolved from production of anti-idiotype antibodies
directed against idiotypic determinants expressed on clonotypic B- and T-cell surface antigen receptors in hematologic
malignancies.2""2MHowever, recent recognition that intracellular proteins can be presented as peptides by class I MHC
molecules suggests a number of alternative sources of tumor
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
321 1
PIVOTAL ROLE OF B7
A) Tumor Cells as APC: Common defects
~~
T Cell Response
Description
Fig 6. This figure depicts the
potential defects that
might
limit the ability of neoplastic
cells t o function as APCs. It also
suggests the functional consequences of tumor-cell:T-cell interactionswith reapect t o potential of each tumor cell as
depicted to elicit an antigen-speciiic T-cell response. In (A), failure of antigen presentation or
absence of necessary cell surface
structures such as MHC and B7
leads to failure of antigen recog
nition; thaefore, noT-cellresponse to antigen is evoked. In
contrast, only the absence ofB7mediated costitnulation is predicted to result in tumor-specific
anergy. (B) Illustrates potential
mechanisms by which tumor
cells may be modified to improve their capacity to serve as
APCt. These modifications include modulation of both tumor
cell surface and function by cytokine treatment,transfection,
or cell fusion.Finally, (C)illustrates the alternative strategy of
using professional APCs to present tumor antigens.
Antigen
Recognition
Proliferation
Cytolytic
Function
Anergy
-
B) Tumor Cells as APC: Approaches to repair defects
antigens (Table 1).30.36,41,l~3.lSS.186
The ability of various tumor
types to process, transport, and present such antigens in the
context of MHC is presently under study; therefore, whether
one or more of these peptide antigens is immunogenic in
vivo is still to be determined.
Finally, the ability of neoplastic cells to provide requisite
costimulatory signals will determine whether a tumor-specific immune response is generated or, alternatively, whether
tumor antigen-specific anergy is induced. Few, if any, solid
tumors constitutively express either the B7 family of costimulatory molecules forming the focus of this review or
+
+
+
+
+
+
+
+
+
-
other costimulatory molecules such as LFA-3 and CD40. In
contrast, by immunohistochemistry, B7can be found on
manybutnot
all B-cell NHLs and on Reed-Stemberg
cells in Hodgkin’s disease tissue sections and cell
~ines.l16,128,187,1S8,190,191T-cell NHLs and circulating B-cell tumors rarely appear to express measurable amounts of
B7.1l4.l16.128 Potential in vivo induction of B7 expression on
neoplasms secondary to systemic inflammatory states or local lymphocyte infiltration has not been studied.
Generation of antitumor immunity in vivo. Inlight of
these potential deficits in tumor cells as APCs, a number of
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
3272
GUINAN ET AL
Table 1. Examples of Tumor Antigens That Are Capable of Eliciting T-cell Responses
~
Where
Potential
Example
Tumor Antigen
Activated oncogene products
Position
Mutated
Rearranged
Tumor suppressor gene products
Reactivated embryonic gene products
Viral gene products
ldiotypic
and
epitopes
lg
Malignancies
Human Present in
mutation
12 point
of ~ 2 1 ' ~ '
bcr-ab1
p53 mutations
MAGEl
Human papilloma virus antigens (E6 and E7)
Epstein-Barr
virus
EBNA-1
gene
products
TCR hypervariable
regions
Surface
10% tumors, including sarcomas, carcinomas,
leukemias, multiple myeloma
CML, ALL
50% tumors, inc. breast, myelodysplasia
50% melanomas, 25% breast cancers
Most cervical carcinomas
Hodgkin's, nasopharyngeal
carcinoma
lg' and
B-cell
TCR' lymphoid
T-cell
malignancies
Table adapted from Pard~ll''~ and other
review^.'^^^'^^^'^^
Abbreviations: EBNA, Epstein-Barr virus nuclear antigen; ALL, acute lymphoblastic leukemia.
approaches have been developed to "repair" these defects
(Fig 6Band C). If the defects can be repaired, then the
outcome should be an effective T-cell-mediated immune
response with appropriate antigen recognition, T-cell proliferation, and induction of cytolytic effector T-cell function.
As illustrated in Fig 6B, a number of cytokines are capable
of upregulating surface expression of adhesion, MHC, and
even costimulatory molecules on both normal and malignant
ce~~s~22.24.104.197.198For example, progenitor cells from patients
with chronic myelogenous leukemia (CML) express low to
absent levels of the adhesion molecule LFA-3
Treatment with interferon-a either in vitro or in vivo increases expression of LFA-3 to normal or near-normal levels
and, interestingly, results in partial correction of the failure
of progenitor cells from patients with CML to generate an
autologous T-cell proliferative response. Interferon-y upregulates the expression of class I and I1 MHC as well as some
adhesion and even costimulatory molecules. In vitro treatment with interferon-y results inmarked improvement in
the ability of many cell types to serve as APCs and has also
been shown to decrease tumorigenicity of transformed or
malignant cell
Transfection of MHC molecules into neoplastic cells provides another potential mechanism enabling tumors to better elicit T-cell responses and
present themselves as targets for effector cytotoxic cells.
Although it is not completely clear why some tumors appear
to present antigen in the context of class I MHC while others
present antigen in the context of class 11, there is experimental evidence that absence of MHC antigens may limit the
ability of the cell to function as an effective APC for tumor
antigens.~95.~96,~99.zn4
For example, the murine Sa1 sarcoma
does not express class I1 MHC or B7.2"7 Transfection of
class I1 not only provides the MHC molecules necessary to
present antigen but also provides a signaling pathway that
results in expression of B7 after interaction of the transfected
tumor with antigen-specificT cells. Therefore, naive animals
injected with tumor that has been transfected with syngeneic
class I1 MHC reject the modified tumor while the unmodified
tumor grows and is fatal. Immunization of naive animals
with the modified tumor also provides protection against
subsequent challenge with the original class II-deficient sarcoma, whereas immunization with irradiated, but unmanipulated, tumor does not provide a protective effect. Similar
results are seen after transfection of syngeneic class I into a
class I-negative melanoma.Z02~'"R~Zw
Although little is currently known about the expression of
B7 family members on tumors other than B-cell neoplasias,
several animal models support the relevance of the B7:CD28
pathway in the generation of a tumor-specific immune response. Murine melanoma or sarcoma lines that did not express B7 were identified and transfected with the B7 gene
and expression of B7 at the cell surface was demonstrated.
In several different murine models, inoculation of animals
with the transfected, B7-positive tumor cells resulted in antigen-specific, MHC-restricted tumor cell rejection in naive
hosts in contrast to fatal tumor growth in control animals
receiving unmodified (B7-negative) cells.202~207~2'o
These models also provide support for the two signal hypothesis with respect to generation of an adequate immune
response. In some cases, CD8' T cells mediated tumor cell
rejection, whereas CD4' T cells did so in others. In the
CD4+ T-cell-mediated model, transfection of B7 into class
I1 MHC-negative tumor cells did not enhance the ability of
the tumor cell to elicit an immune response and, consequently, tumor growth was notim~aired."~
However, rendering tumor cells both class I1 MHC and B7-positive in the
same model resulted in ability to induce a vigorous antitumor
effect (as in Fig 6B). Therefore, if tumor cells are to function
as APCs and generate an efficient tumor-specific T-cell response, they need to be able to deliver both signal 1 and 2.
Further support for this notion is found in similar experiments in which tumors (derived from 2 T-cell NHLs, a mastocytoma and a melanoma) were also rejected after transfectionwithB7
although other tumors (3 sarcomas and a
melanoma) continued to grow aggressively even after B7
transfection and cell surface expre~sion.~'~
Although all 8 of
the unmodified tumors grew in naive hosts, only the tumors
with evidence of immunogenicity before B7 transfection (as
assessed in the immunization and rechallenge experiments
described above) were capable of eliciting an antigen-specific immune response after being rendered B7 cell surface
positive. Again, the most straightforward explanation of
these results is that the immunogenic tumors were capable
of providing signal 1 to T cells. Transfection with B7 rendered them capable of providing signal 2, thus enabling the
tumor cell to function as an APC competent to generate a
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PIVOTAL ROLE OF B7
full, antigen-specific immune response (Fig 6B). That B7
expression did not enhance the ability of the nonimmunogenic tumors to elicit a T-cell response, on the other hand,
might be presumed to reflect defects in providing signal
1. Interestingly, in these latter experiments (in which the
antitumor effect appeared to be mediated by CD8+ T cells),
3 of 4 nonimmunogenic tumors studied lacked class I cell
surface antigens, suggesting at least one reason for failure
of the presentatiodrecognition step. Other mechanisms, such
as lack of recognizable tumor antigens or deficits in processing and transportation of antigens, might also be invoked
to explain the failure of these tumor cells to stimulate an
immune response despite B7 transfection. On the whole,
therefore, these B7 transfection experiments provide significant support for the hypotheses that (1) tumor cells may be
limited in their capacity to function as APCs; ( 2 ) such failure
may lead not only to abortive immune responses but also to
tumor antigen-specific tolerance; and (3) induction of appropriate cell surface molecules, including the costimulatory
ligand B7, on tumor cells may lead to antigen-specific immune responses (tumor rejection) in naive hosts.
Other experimental designs have also shown the ability
of B7 transfection to alter the capacity of neoplastic cells to
elicit protective immunity against tumors. In some experiments, naive animals were inoculated (ie, immunized) with
B7-transfected tumor ce11s.202~207~209
This inoculation led not
only to primary rejection of the modified tumor cells, as in
the experiments described above, but also to a significant
degree of protection against subsequent inoculation with unmodified, B7-negative tumor cells. In these systems, the induction of systemic immunity could be shown to be antigen
specific and was not observed when the transfected tumor
cells were derived from nonimmunogenic tumors.209Equivalent schedules of immunization with equal numbers of unmodified killed or irradiated tumor cells afforded little protective effect. However, repetitive immunization with these
untransfected but immunogenic tumor cells was able to afford considerable protection to subsequent challenge with
nonirradiated tumor. Again, these experiments support the
hypothesis that effective antigen-specific immunity requires
delivery of both signal 1 and 2. Furthermore, they suggest
that this goal may possibly also be accomplished, albeit less
well, by presentation of shed or phagocytosed tumor antigens
by professional, nonneoplastic APCs (Fig 6C). Because inoculation with killed or irradiated tumor may facilitate sufficient presentation of shed or phagocytosed tumor antigen
by resident professional APCs, identification, isolation, and
administration of common tumor-specific antigens may provide an alternative strategy to immunize the host.
Yet another mechanism for generation of a specific tumorprotective response is suggested by the experiments of Guo
et a1’” in which the requisite elements enabling a tumor cell
to function as an APC were established by fusion of the
tumor cell with purified activated syngeneic B cells (Fig
6B). In the reported experiments, the tumor cell, a hepatocarcinoma, expressed limited amounts of MHC class I and 11
and limited (ICAM-1) or absent (LFA-1 and B7) amounts
of adhesion and costimulatory molecules. In contrast, these
molecules were all highly expressed by both the activated
3273
B cells and the hybrid, fusion product of both cells. Inoculation of nude (T-cell-deficient and immunoincompetent)
mice with the hybrid cells resulted in tumor formation showing the retained neoplastic potential of the fused cells. However, immunologically normal animals inoculated with the
hepatoma-B-cell hybrid were able to reject the cells and
remain tumor free. In addition, prior exposure of animals
to the hybrid cells rendered them resistant to subsequent
challenge withunmodified hepatoma, ie, the hybrid cells
were able to immunize naive animals. In these experiments,
both helper and cytotoxic T cells were essential for the generation of the antigen-specific antitumor cell response. Thus,
rendering the neoplastic cell capable of delivering both signal l and signal 2 to antigen-specific T cells by either transfection of relevant molecules or cell fusion with professional
APCs that express requisite molecules can result in generation of effective and tumor-specific immunity after primary
immunization.
A major limitation of all the above model systems is that
modified tumor cells are injected into naive, but not tumorbearing, hosts. For clinical translational experimentation, the
more relevant model would examine the ability of modified
tumor cells to influence the growth of an established tumor
or minimal residual disease. In a melanoma model in which
tumor inoculation universally leads to disseminated metastatic disease and death, intravenous injection of B7transfected cells 4 days after inoculation of B7-negative
tumors resulted in prolonged survival inall animals and
long-term survival in nearly half.*” Similarly, in the hybrid
B-cell-hepatoma model, either intrahepatic injection or intrahepatic implantation of tumor followed by subcutaneous
injection of hybrid cells 10 days later was associated with
80% to 100%tumor-free survival.’” Although these studies
are still far removed from treatment of patients with malignant disease, they provide proofof concept that in some
tumor-bearing hosts itmaybe
possible to induce tumorspecific immunity.
Taken together, these experiments suggest that manipulation of host T-cell response to malignant cells to immunize
against or treat tumors may be possible. If certain antigens
were common to a sufficiently broad spectrum of malignancies, routine vaccination with either a tumor-APC hybrid or
transfected tumor cell might be possible. Possibly, a multivalent vaccine comprising the most frequently occurring malignancies for a given age and sex cohort could be used. Alternatively, patients at identified high-risk for particular
malignancies (eg, womenwith p53 mutations and family
histories of breast cancer) could be selectively immunized.
Efficacy of any given approach is predicated on the ability
to overcome possible pre-existing T-cell tolerance to the
established tumor. Inother words, in vivo therapy with tumor
cells that have had their “deficiencies” as APCs remedied
ex vivo by cell fusion or transfection may be efficacious
but only if pre-existing tolerance does not exist or can be
abrogated. In vivo therapy with cytokines capable of inducing neoplasms to express molecules important in immunogenicity may also be efficacious either independently or as
adjunctive therapy. Theoretical approaches that might circumvent pre-existing tolerance in the tumor-bearing host
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
3214
include ex vivo expansion of sorted, naive host T cells stimulated with tumor transfectants or hybridomas as well as administration of similarly expanded and targeted cells derived
from an HLA-matched sibling or, potentially, alternative donor. In vivo therapy directed against mature, tolerant T cells
followed by infusion of large numbers of immunogenic, manipulated tumor cells might also generate sufficient specific
activity in emerging, naive cells.
GUINAN ET AL
the B7:CD28 pathway in vivo with either antimurine B7-2
MoAb or CTLA4-Ig can prevent the development of experimental allergic encephalitis, the murine model for multiple
sclerosis.""
In
aggregate, these studies implicate the
B7:CD28 pathway in the pathogenesis of autoimmune diseases and perhaps also those with a significant reactive component. They also provide reasonably compelling evidence
that modulation of the pathway may have a beneficial effect.
Although more speculative, inappropriate B7-mediated coPOTENTIAL FOR CLINICAL EXPERIMENTATION:
stimulation may also play a role in the pathogenesis of autoA PERSPECTIVE
immune cytopenias and even a subset of aplastic anemia.'?'
Considering the well-established, albeit poorly predictable,
Therapeutic reagents intended to modulate the B7:CD28
efficacy of anti-T-cell antisera and other immunosupprescostimulatory pathway have yet to enter the clinical arena.
sive medications in treating these conditions, it would not be
However, because several teams of investigators are conteinappropriate to undertake clinical trials to block B7:CD28mplating initiation of clinical trials, it is of more than acamediated costimulation. Certainly, a subgroup of patients
demic interest to discuss which hematologic diseases might
with aplastic anemia respond to antithymocyte globulin
provide the promising clinical settings for such studies.
and, in fact, some of these patients are cured by this apImmunosuppression. The greatest therapeutic potential
p r o a ~ h .Antibodies
~ ~ ~ - ~ that
~ ~inhibit the B7:CD28 andpotenarising from modulation of the B7:CD28 pathway resides in
tially
other
costimulatory
pathways could be amongthe
the induction of immunosuppression. Reagents that commajor effective reagents in these heteroantisera. Because
pletely block the B7:CD28 pathway will likely be most effimodulation of this pathway has been shown to be effective
cient in inducing significant immunosuppression (Fig 5).
in both tolerizing previously sensitized cells and downreguVirtually all preclinical in vivo studies have used CTLA4-Ig,
lating specific antibody production, modulators of this pathwhich has been extremely effective in blocking B7 familyway may also prove useful intreatment of antibody-mediated
mediated costimulation.'72"7s Anti-CD28Fab MoAb also
inhibitor states (eg, acquired coagulopathies) or even in treatcompletely blocks costimulation delivered by B7 family
ment or prevention of transfusional allosensitization.''5
members and may have equivalent potential to be an effecAnother fertile area for investigation is allogeneic BMT
tive immunosuppressive
Blocking of onemore
or
in which graft failure, GVHD, treatment-related toxicity, opmembers of the B7 family with specific MoAbs may potenportunistic infection, and B-cell lymphoproliferative disease
tially suppress B7-mediated costimulation, although precliniremain significant problems. Current attempts to increase the
cal studies supporting this hypothesis have yet tobe reported.
size of the donor pool by using mismatched family or unreFurther, administration of cytokines such as IL-10 that downlated BM donors serve to increase both the incidence and
regulate B7 expression or, alternatively, blocking signaling
severity of these complications.'2"23xIn these settings, inducby cytokines such as IL-7 or surface structures such as CD40
that upregulate B7 expression may also be u~eful."'~~''~''~tion of host tolerance to donor-specific alloantigens might
facilitate engraftment and reduce the incidence of graft rejecHowever, entry level clinical studies will likely involve
tion, whereas induction of donor tolerance to host-specific
blockade of B7:CD28 using CTLACIg or, potentially, solualloantigens might provide a new, more specific approach to
ble CTLA-4.
treatment or prevention of GVHD.'7'.''9 Modulation of the
For the general hematologist, the ability to induce antigenB7:CD28
pathway is most efficient in preventing immune
specific immunosuppression would potentially facilitate the
responses by both naive and previously sensitized cells and
treatment of a broad range of disorders in which either autoless markedin its ability to turnoff established effector
immune or alloimmune responses produce hematologic dysfunction. For example, the B7:CD28pathwayisinvolved
function. Increasing evidence suggests that a number of autoin the generation of cytolysis but not in cytolytic effector
immune diseases can be prevented or suppressed by
functi~n.~~
This
" ~ ~finding
~'
suggests that it is unlikely that
modulating the B7:CD28 p a t h ~ a y . ~ For
~ ~ -example,
~''
expersignificant clinical efficacy would be demonstrated.
iments in transgenic who constitutively express susceptibilConsiderably greater potential rests in the prevention of
ity genes for diabetes and the B7 gene in pancreatic islet
GVHD. One strategy would consist of attempts to specificells suggest that inappropriate expression of B7-1 costimucally anergize alloreactive T cells in the donor BM to host
lation may be involved in the pathogenesis of this disease.?"
alloantigens. Tolerizing the mature donor T cell would in
In a second transgenic murine model, only coexpression of
effect
reproduce the same functional phenotype as depletion
high levels of both B7-1 and class I1 MHC antigens resulted
of mature T cells from donor BM withrespect to the immediin autoimmune destruction of islet cells, whereas expression
ate inability or limited capability of the graft to induce
of either molecule alone was insufficient to overcome in vivo
GVHD. However, T cells tolerant to specific alloantigens
t o l e r a n ~ e .One
~ ' ~ might postulate that either the susceptibility
would retain function against viral and other opportunistic
genes or the increased expression of MHC result in presentainfections, whereas this benefit is lost with global T depletion of an antigen which can be seen by autologous T cells
ti~n.'~'In addition, the increased incidence of both graft
as foreign (signal l), whereas expression of B7-1 enables
rejection or failure, relapse, and B-cell lymphoproliferative
delivery of signal 2 and evocation ofan anti-islet T-cell
disease observed after transplantation of T-cell-depleted
response. Recent studies in mice also show that blockade of
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
PIVOTAL ROLE OF B7
3215
BM might wellbe mitigated.227-22y.232-2383N3
Advantages of
geneity has obvious implications for the generation of an
antigen-specific antitumor response. Whether morphologic,
this approach in comparison to immunosuppressive therapies
arise from the fact that immunosuppressive medications are,
phenotypic, andor genetic heterogeneity impacts on the antiby definition, general in their effects, contributing to many
genicity of a tumor is presently unknown.'" Additional conof the problems cited above. These drugs also have significerns about the applicability of the approaches discussed
cant organ toxicities. Limitations to this approach include
here arise from the observation that, although some hematologic malignancies are the neoplastic counterparts of APCs
selection of appropriate host tissues that present the antigens
and some express B7 family members, these tumors are not
necessary to generate tolerance to the classic GVHD targets
of skin, bowel, and liver.'78*23y
A related concern is whether
rejected. The resolution of this apparent paradox may lie in
considering the evolution of the malignant clone. For examanergy induced in vitro will be long lived or reversed in
vivo. However, even a limited period of tolerance might
ple, Ig gene rearrangements and bcl-2 translocations can be
found in the earliest pre-B cells at which stage both normal
have significant advantages over currently available technoand neoplastic pre-B cells express class I and class I1 MHC
logies and approaches.
but not B7.188~190~193
Although the Reed-Sternberg cells of
Another approach would involve the in vivo administration of reagents to block the B7:CD28 pathway. As was
Hodgkin's disease are strongly B7 positive, the clonogenic
cell in Hodgkin's disease is ~ n k n o w n . ' ~Therefore,
~ ~ ' ~ ~ in
shown in the murine model of allogeneic cardiac transplantalymphohematopoietic malignancies, one could imagine that
tion, intravenous administration of CTLA4-Ig can result in
tumor-specific tolerance maybe generated very early by
immunosuppression or even induction of tolerance to alloantigen.173.174
Importantly, in those studies, the timing of intratumors capable of delivering signal l butnot signal 2.244
venous CTLA4-Ig administration relative to the antigenic
Attention to mechanisms by which antigen-specific tumorexposure and the mechanism (timing, dose, and site) by
cell tolerance might be reversed will be critical to the clinical
which the foreign antigens were introduced into the host
efficacy of this approach.
had significant impact on the success of the intervention.
A long and unsuccessful history has accompanied attempts
Preliminary studies in mice suggest that intravenous adminto develop tumor-cell vaccines to treat tumor-bearing
hosts~~82,~84-186,2~
istration of CTLA4-Ig can significantly reduce but not elimiTumor cell vaccines have consisted of einate lethal GVHD in fully MHC-mismatched mice.'75As in
ther modified whole tumor cells or alternatively extracts dethe cardiac transplant model, refinements in the experimental
rived from tumor cells. We will not attempt to review this
protocol may improve these results. Alternatively, a combiextensive literature here, but rather briefly propose examples
nation of in vitro and in vivo therapy may be more effective
of alternative strategies based on manipulation of costimulaor some combination of B7:CD28 blockade and inhibition
tory molecules. The success of prophylactic vaccination deof effector cell function (eg, anticytokine antibody) may be
pends on generation of sufficient numbers of long-lived cytonecessary. Additional concerns regarding maintenance of anlytic T cells to provide immunologic surveillance.
ergy suggest that techniques resulting in clonal deletion of
Unfortunately, it appears unlikely that mosthematologic madonor alloreactive T cells might provide an even more effeclignancies will be amenable to such an approach. Unlike
tive and potentially less reversible approach.
candidate tumor-associated antigens on human solid tumors
Immunostimulation. Although
modulation
of the
(Table l), fusion proteins from known translocations comB7:CD28 pathway provides a potential mechanism for inmon to hematologic malignancies are characterized by sufducing tumor-specific immunity, one can readily foresee a
ficient heterogeneity that a common candidate tumor antigen
number of obstacles to the implementation of this approach.
has yet to be identified. If a candidate antigen were discovIn addition to the specific issues of tumor antigenicity deered, gene transfer strategies capitalizing on the function of
tailed above, central concerns include ( l ) existence of a sufcostimulatory molecules might be implemented. Genes for
ficient number of antigen-specific precursor T cells, (2) stothe tumor antigen as well as genes for adhesion and costimuchastic issues related to tumor cell burden, (3) the ability of
latory molecules could be simultaneously transfected into
specific class I and class I1 molecules to present each and
selected host tissues. For example, vaccinia virus packages
every peptide antigen, and (4) tumor-cell heterogeneity
could be used to infect skin with both tumor antigen and
within a given malignancy. With respect to the T cells, not
B7. Alternatively, naked DNA encoding both tumor antigen
only may there be an inherent limitation in the number of
and costimulatory molecules could be injected into muscle
cytotoxic and helper T-cell precursors capable of recognizing
leading to expression of the encoded proteins by the "inand responding to a specific malignancy, but conventional
fected" muscle cells. These approaches have been promising
chemoradiotherapy may further limit the T-cell repertoire by
in murine systems. Failure of any of these approaches might
depleting both specific and nonspecific T-cell populations.
be the result of any of the general concerns listed above as
Of course, absolute tumor burden and growth fraction may
well as failure of the immune system to maintain sufficient
exceed even the most potent cytolytic T-cell antitumor relong-term memory.
sponse. In addition, certain tumor peptide antigens may not
For the tumor-bearing host, two treatment strategies will
be efficiently presented to antigen-specific T-cell precursors
be considered, although many variations are possible. The
by the patient's unique MHC m o l e c ~ l e s ~ 'Therefore,
~ ' ~ ~ ~ ' ~ ~ first approach, termed adoptive transfer, would entail in vitro
failure to generate a T-cell antitumor response might be
isolation and expansion of tumor-specific cytolytic T lyminherent in the patient's genetic immunologic repertoire.
p h o c y t e ~ . ' ~ ~Currently,
" ~ ' . ~ ~ isolation, expansion and adminWith respect to the malignancy itself, tumor-cell heteroistration of T-cell tumor-infiltrating lymphocytes (TIL) is
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
3276
GUINAN ET AL
limited to tumors in which sufficient T-cell infiltrate exists
such that isolation of cells is practical. This limitation is
particularly pertinent for tumors of the lymphohematopoietic
system. The proposed strategy differs in that it would not
rely on expansion of T cells previously sensitized in vivo
but would also attempt to generate and expand additional
specific cytolytic T cells from antigen-specific precursors.
Isolated, purified leukemia or lymphoma cells could be used
as APCs to stimulate this population. Tumor cells could be
modified as described in preclinical animal models (Fig 6B)
to facilitate adhesion, antigen recognition, and costimulation
and improve the capacity of the malignant cells to serve as
immunogens. Alternatively, tumor-specific peptide antigen
could be presented to T cells by professional APCs that
might generate a much moreefficient and effective antitumor
response (Fig 6C). The success of both approaches is dependent on the ability of T cells to respond to the antigen;
therefore, the T-cell repertoire mustbe intact. If T cells
have been tolerized to the tumor in vivo, tolerance must be
reversed in vitro to enable induction and expansion of antitumor-specific T cells. Manipulation of the B7:CD28 a n d
or other costimulatory pathways provides a potential avenue
for reversal of anergy.
SUMMARY
The above story illustrates the translation of basic scientific discoveries to the clinic. In vitro and preclinical in vivo
experimentation suggests that modulation of the B7:CD28
pathway will result in either amplification or suppression of
the immune response. Considering the frequency with which
diseases characterized by either inadequate or dysregulated
immune function present to the practicing hematologist or
oncologist, it is not difficult to envisage clinical applications
for reagents that modulate this pathway. However, we still
have much to learn about the function and clinical potential
of this and other potentially redundant costimulatory pathways and therefore we suspect that this story will become
considerably more complex over the next few years.
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Pivotal role of the B7:CD28 pathway in transplantation tolerance and
tumor immunity
EC Guinan, JG Gribben, VA Boussiotis, GJ Freeman and LM Nadler
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