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TRANSPLANTATION
From the Department of Adult Oncology, Dana Farber
Cancer Institute, and the Division of Medical Oncology, Brigham and
Women's Hospital, Harvard Medical School, Boston, MA.
Blockade of B7/CD28 costimulation allows human haploidentical bone
marrow transplantation without graft-versus-host disease. This study
shows that blockade of B7/CD28 in anergizing mixed lymphocyte reaction
(MLR) cultures of peripheral blood mononuclear cells results in the
generation of alternatively activated macrophages (AAM The success of allogeneic bone marrow
transplantation (ABMT) is limited by the restricted donor pool and the
morbidity of graft-versus-host disease (GVHD). Donor T cells present in
the graft are mediators of GVHD. These donor T cells recognize host alloantigens and initiate a cascade of pathophysiologic events, which
result in the development of the clinical syndrome of
GVHD.1 Although GVHD can be prevented or ameliorated by
nonspecific immunosuppression or T-cell depletion, these
approaches are associated with increased infection and relapse rates
and, in the case of T-cell depletion, with increased graft failure
and lymphoproliferative disease.2
Studies in murine and subhuman primate models3-7 and more
recently a clinical trial in humans8 have provided great
promise that induction of T-cell anergy by blockade of costimulation
may prevent GVHD without nonspecific immunosuppression or T-cell
depletion. Anergy in vitro and its in vivo counterpart, tolerance, are
defined as the inability of viable, antigen-specific T cells to produce interleukin-2 (IL-2) or clonally expand when rechallenged with fully
competent antigen-presenting cells (APCs) delivering T-cell receptor
(TCR) and costimulatory signals.9 Anergy can be
induced when T cells are stimulated via TCR without costimulation or
IL-2.9,10 The B7-1/B7-2:CD28 pathway is the dominant
costimulatory pathway and has a critical role in the regulation of
T-cell viability, cytokine production, clonal expansion, and effector
function.11 The in vivo biologic significance and the
clinical relevance of this pathway have been validated in multiple
models for allogeneic renal, cardiac, and bone marrow
transplantation.3-5 We have shown that ex vivo blockade of
this pathway can inactivate the ability of donor haploidentical T cells
to recognize host alloantigens, thereby significantly reducing the
incidence of GVHD in patients undergoing haploidentical bone marrow
transplantation.8
T lymphocytes are the mediators of immunity, but their function is
under the control of APCs. Monocytes, the most abundant APCs, develop
from hematopoietic precursors, enter the bloodstream, and follow
divergent differentiation programs to form the wide variety of
morphologically and functionally distinct macrophages. The activation
and differentiation program of monocytes into macrophages is determined
by the presence of specific cytokines in selected microenvironments in
vivo or in culture supernatants in vitro. The balanced
macrophage-activation hypothesis states that in parallel with the
Th1/Th2 paradigm, macrophages can be divided into "classically" activated (CAM In our recent clinical study, histoincompatible bone marrow grafts
infused to patients after ex vivo blockade of the B7/CD28 pathway with
cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig contained donor T
cells that exceeded by 1 or 2 orders of magnitude the suggested
threshold dose for minimizing GVHD.8 These patients had
additional high-risk factors for GVHD, including mismatching of the
donor and recipient, recurrent or persistent disease, irradiation-based conditioning, and cytomegalovirus positivity.8 Strikingly, the incidence of GVHD by this treatment approach not only was not
increased, but was significantly reduced as compared with that observed
after classic treatments. To determine whether besides inhibition of
CD28 signals, additional mechanisms may contribute to the
immunosuppression of host-specific donor T cells by this treatment
approach, we examined the immunophenotypic, molecular, and functional
properties of macrophages present in anergizing mixed lymphocyte
reaction (MLR) cultures of peripheral blood mononuclear cells (PBMCs).
Here we show that blockade of the B7/CD28 pathway results in the
generation of AAM Mixed lymphocyte reactions
Immunofluorescence and flow cytometry
Carboxyfluorescein diacetate succinimidyl ester labeling and analysis Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR) and was reconstituted and used according to the manufacturer's protocol. Briefly, CFSE was diluted to a final concentration of 1 µM, and cells at a concentration of 107/mL were incubated at 37°C for 15 minutes in nonsupplemented RPMI (Gibco-BRL). The labeled cells were washed once with phosphate-buffered saline (PBS) containing 10% FCS, washed 3 times with PBS, and set to the indicated final concentration before initiation of MLR cultures.FITC-dextran uptake Uptake of FITC-dextran (molecular weight 70 000; Sigma, St Louis, MO) was evaluated as described previously.32 Briefly, CD14+ cells isolated from the indicated cultures were resuspended at 1 × 106 cells/mL in RPMI containing 10% FCS and 25 mM HEPES and incubated with FITC-dextran (1 mg/mL) for the indicated time intervals. The quantitative uptake of FITC-dextran was determined by flow cytometry.Cytokine enzyme-linked immunosorbent assay Culture supernatants were harvested at various time intervals of primary and secondary cultures and analyzed by enzyme-linked immunosorbent assay (ELISA) for cytokine levels of IL-2, IFN- , IL-4,
IL-10, and TGF- (R&D Systems). Assessment of total NO was done by
colorimetric enzymatic assay according to the manufacturer's protocol
(R&D Systems).
Reverse transcription-polymerase chain reaction Before RNA extraction, CD4+ and CD14+ cells isolated from MLR cultures by positive selection using anti-CD4 or -CD14 MicroBeads and MACS columns (Miltenyi Biotec), RNA was prepared by the RNAsol kit (Tel-Test, Friendswood, TX), and 2 µg RNA was used for reverse transcription as described previously.33 Polymerase chain reaction (PCR) amplification of cDNA was performed using specific oligonucleotides for IL-10, 5'-AAGCTGAGAACCAAGACCCAGACATCAAGGCG-3' (forward) and 5'-AGCTATCCCAGAG CCCAGATCCGATTTTGG-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5'-GTGAAGGTCGGAGTCAACG-3' (forward) and 5'-ACCAGGAAATGAGCTTGACAAA-3' (reverse); and AMAC-1, 5'-AAGCCCCAGCTCACTCTGAC-3' (forward) and 5'-ACCTGGCTTGGGGCACT-3' (reverse). Fifteen microliters of each of the final reaction products was analyzed by electrophoresis on 2% agarose gel containing ethidium bromide.Suppression subtractive hybridization Suppression subtractive hybridization34 was done with the use of the PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer's protocol. Briefly, cDNA from CD14+ cells isolated from anergizing cultures was used as "tester" (in which differential gene expression is searched), and cDNA from CD14+ cells isolated from priming cultures was used as "driver" (to which gene expression is compared). Tester cDNA was ligated to 2 different cDNA adaptors, tester and driver cDNAs were hybridized twice, and hybrid sequences were removed. Consequently, the remaining unhybridized cDNAs represented genes that are expressed in the tester but are absent from the driver mRNA. Subsequently, only unhybridized molecules containing adaptor sequences (and therefore originating from tester cDNA) were exponentially amplified by PCR using adaptor-specific primers. After a subtracted cDNA library was obtained, confirmation that individual clones were indeed differentially expressed was done by differential screening using the PCR-select differential screening kit (Clontech), as indicated by the manufacturer's protocol.
Blockade of B7/CD28 costimulation results in the generation of
macrophages with immunophenotypic markers of AAM To determine whether blockade of the B7/CD28 pathway with either
anti-B7 mAbs or CTLA4-Ig influenced the maturation and differentiation program of monocytes present in the anergizing cultures, we first examined surface markers that had been previously determined to be
differentially expressed in classically and alternatively activated macrophages.13,14 We examined the expression of CD14, MHC
class II, CD23, CD32, CD64, CD16, and CD163. At 48 hours of culture, there was a detectable difference in the expression of CD14 but not in
the expression of any of the other markers (Figure
1, top panel; and data not shown). At
days 5 and 7, a significantly enhanced expression of CD14 and MHC class
II was observed on monocytes isolated from cultures treated with
anti-B7 mAbs as compared with monocytes isolated from cultures with
medium alone (Figure 1, top and middle panels) or with control mAbs
(data not shown). Similar results were observed when CTLA4-Ig was used
instead of the combination of anti-B7-1 plus anti-B7-2 mAbs to induce
anergy (data not shown). Further analysis of markers expressed on
CD14+ cells in each population revealed that
CD14+ cells isolated from anergizing cultures treated with
either the combination of anti-B7-1 plus anti-B7-2 mAbs or with
CTLA4-Ig had increased expression of CD23 (Figure 1, lower panel; and
data not shown). The expression levels of CD64 and CD32 were only
slightly lower on CD14+ cells isolated from anergizing
cultures than on CD14+ cells isolated from cultures with
medium alone (data not shown). In contrast to previous
reports,18 no detectable differences were observed in the
expression of CD16 and CD163 on the CD14+ cells in the
different culture conditions (data not shown).
Because B7-1 (CD80) is induced and the very low constitutive levels of B7-2 (CD86) are up-regulated on monocytes after activation,36,37 we examined whether CD14+ cells isolated from the anergizing and priming cultures had different expression of B7-1 or B7-2. At all time intervals of culture tested, equivalent levels of B7-1 and B7-2 were induced on CD14+ cells in the anergizing and priming cultures (data not shown). Analysis of the dendritic cell-specific marker CD83 showed no detectable levels of CD83 expression in our cultures at any time point tested (data not shown). This is most likely due to the specific culture requirements for dendritic cell growth, which were not established in these classic MLR cultures. Distinct cytokine profile in anergizing cultures contributes to the
generation of AAM  were detected in
CD14+ cells isolated from anergizing MLR cultures. Previous
studies have suggested that the differentiation program of macrophages is significantly influenced by cytokines present in distinct
microenvironments in vivo or in cultures in
vitro.13,14,32,38,39 Specifically, it has been shown that
classic activation of macrophages is induced by proinflammatory
cytokines such as IFN-, whereas alternative activation of
macrophages is induced by anti-inflammatory cytokines such as IL-4 and
IL-10.13,14 It is well documented that the B7/CD28
costimulatory pathway dramatically augments cytokine production by T
cells, and blockade of this pathway leads to inhibition of cytokine
production.40 Moreover, studies in a different
experimental system have shown that although production of IL-2 and
IFN- is blocked in anergizing MLR cultures, production of IL-10 is
augmented.41 Therefore, we examined the expression of
cytokines at various time intervals of our MLR cultures. Because the
functional characteristics of anergic cells are identified on
rechallenge, expression of cytokines was also assessed during
rechallenge (secondary) cultures. Blockade of the B7/CD28 pathway with
the combination of anti-B7-1 plus anti-B7-2 mAbs during primary MLR
resulted in dramatic inhibition of IL-2, IL-4, and IFN- production
in both primary and secondary cultures (Figure
2A,B). In contrast, addition of
anti-B7-1 plus anti-B7-2 mAbs during primary MLR resulted in
increased production of IL-10. Although there was only a slight
increase in IL-10 levels during primary culture in the presence of
anti-B7-1 plus anti-B7-2 mAbs (Figure 2A), there was a dramatic
increase in IL-10 levels in rechallenge, secondary cultures of cells
that had been treated with anti-B7 mAbs during primary MLR (Figure 2B).
TGF- was not detected in any of the conditions of primary or
secondary MLR cultures (data not shown). The observation that IL-4
production was inhibited by blockade of B7-mediated costimulation with
anti-B7 mAbs provides an explanation for the lack of significant
down-regulation of CD32 and CD64 on macrophages isolated from
anergizing cultures. IL-4 is the critical factor that mediates
down-regulation of CD32 and CD64, and this effect is counteracted by
IFN-.21 Therefore, the presence of IFN- in
priming cultures in medium prevents IL-4-mediated down-regulation of
these receptors, whereas in anergizing cultures, none of these
cytokines is present to mediate an effect.
It has been proposed that IL-10 leads to the generation of
AAM
AMAC-1 is a cytokine closely related to MIP-1 AAM were detected in CD14+ cells isolated
from anergizing MLR cultures. Previous studies have shown that although
AAM have reduced capacity to process antigens, they have increased
phagocytotic activity.32,38 To determine whether
macrophages isolated from anergizing cultures had gained this
functional feature of AAM, we examined their phagocytotic ability.
CD14+ cells were isolated by positive selection from viable
responder PBMCs and were incubated with FITC-dextran for various time
intervals. Subsequently, endocytotic capacity of macrophages was
determined by flow cytometry. As shown in Figure
4A, macrophages isolated from cultures
treated with anti-B7-1 plus anti-B7-2 mAbs showed 4 times greater
endocytosis of FITC-dextran as compared with cells isolated from
priming cultures in medium alone or with control mAb (data not shown).
Increased phagocytotic capacity was also observed when CTLA4-Ig was
used instead of anti-B7-1 plus anti-B7-2 mAbs in anergizing MLR
cultures (data not shown).
Intracellular processing of phagocytosed antigen requires enzymatic
activation of nitric oxide reductase, which subsequently generates NO
and O2.42 It has been shown that although
AAM To determine the in vivo biologic significance of these findings, we examined whether macrophages in priming and anergizing culture conditions might have differential phagocytotic ability against cell populations in the MLR culture. CFSE-labeled responder PBMCs were cultured with unlabeled allogeneic stimulators. This approach allowed us to discriminate these 2 populations and to follow the fate of each one during culture. Strikingly, culture in the presence of anti-B7-1 plus anti-B7-2 mAbs or with CTLA4-Ig resulted in a significant decrease of the unlabeled (CFSE-negative) stimulator cells as compared with those detected in cultures with medium alone (Figure 4C). This event became detectable after 3 days of culture and was further augmented by day 7. This observation indicates that a higher degree of scavenging and clearance of the unlabeled (CFSE-negative) stimulator cells occurred in anergizing culture conditions. As mentioned earlier, previous studies have shown that AAM IL-10 has an active role in the generation of AAM .13,32 To examine whether IL-10 might be responsible for the enhanced phagocytotic activity observed in macrophages isolated from anergizing cultures, we added
anti-IL-10 neutralizing mAb in anergizing MLR cultures treated with
anti-B7-1 plus anti-B7-2 mAbs. Culture under these conditions significantly reduced the phagocytotic capacity of macrophages as
determined by FITC-dextran uptake (Figure 4A). Moreover, anti-IL-10 neutralizing mAb increased the levels of NO produced in the culture treated with anti-B7-1 plus anti-B7-2 mAbs (Figure 4B). Thus, IL-10
mediates at least one mechanism involved in the generation of
macrophages with increased phagocytotic capacity in anergizing MLR
cultures. Taken together, all of these results strongly suggest that
during induction of T-cell anergy by blockade of the B7/CD28 pathway, a
distinct activation program is initiated in macrophages, leading to
immunophenotypic and functional properties of AAM.
Differential gene expression in macrophages isolated from anergizing and priming cultures To examine whether AAM that were generated in anergizing MLR
cultures had differential expression of other macrophage-specific genes
as compared with CAM generated in priming cultures, we performed
suppression subtractive hybridization using PCR-select cDNA
subtraction. RNA was extracted from CD14+ cell populations
isolated from anergizing and priming cultures, and cDNA was prepared by
RT-PCR. cDNA from cells isolated from anergizing cultures was used as
"tester" and cDNA from priming cultures was used as "driver"
cDNA, and suppression subtractive hybridization was performed as
described in "Materials and methods."34 After a
subtracted cDNA library was obtained, confirmation that individual
clones were indeed differentially expressed was done by differential
screening, using as probes either cDNA prepared from anergizing
cultures (forward probe) or cDNA prepared from priming cultures
(reverse probe). This approach allows the identification of genes in
the subtracted library that are selectively detected by the forward
probe and therefore are selectively expressed in the cDNA population
isolated from anergizing cultures.
Among 200 recombinant clones tested, 11 clones represented genes that
were selectively or differentially expressed in macrophages isolated
from anergizing cultures (data not shown). Here, we present a number of
genes related to previously determined functional properties on
macrophages. Two receptors with known scavenging activity were
selectively expressed on macrophages isolated from anergizing cultures:
macrophage mannose receptor and sortilin. Macrophage mannose receptor was previously identified as a marker of
alternative immunologic macrophage activation.15 This is an important phagocytotic receptor mediating binding and ingestion of
microorganisms with surface mannose residues and soluble
mannose-containing glycoproteins. In fact, it has been reported that
macrophage mannose receptor has the most important role in mediating
endocytosis and scavenging by macrophages. It is expressed in alveolar
macrophages, and its expression is increased by steroids and inhibited
by IFN- Besides these 2 receptors, which are directly linked to the phagocytic
and scavenging properties of macrophages, a number of genes previously
associated with the maturation and differentiation program of monocytes
were differentially expressed between macrophages isolated from
anergizing and from priming MLR cultures. These results provide molecular evidence that distinct activation and differentiation gene programs are initiated on macrophages during anergizing and priming culture conditions. These distinct gene programs result in distinct functional properties of macrophages. Macrophages isolated from anergizing MLR cultures mediate suppression of T-cell responses Previous studies have shown that APCs generated in the presence of IL-10 do not induce T-cell responses but are rather immunosuppressive.25,32,38,39 To examine whether AAM
generated in anergizing cultures have immunosuppressive properties, we
isolated CD14+ cells from anergizing and priming cultures
by positive selection. These CD14+ cells were subsequently
added in a new primary MLR culture and in a secondary MLR culture of
purified CD4+ original responder cells and original PBMC
stimulators. CD14+ cells isolated from anergizing primary
cultures, which have phenotypic, functional, and molecular properties
of AAM, reduced alloresponses of CD4+ original responder
cells in a new primary MLR culture (Figure 5A) and in a secondary MLR culture
(Figure 5B). This inhibitory effect was dose dependent (data not
shown). Moreover, AAM also reduced alloresponses in primary and
secondary MLR cultures of different responder CD4+ cells
(data not shown). In contrast, CD14+ cells isolated from
priming cultures in medium alone, which have phenotypic and molecular
properties of CAM, augmented primary and secondary MLR responses of
original CD4+ responder cells (Figure 5A,B). These CAM
also augmented primary and secondary responses of different
CD4+ responder cells (data not shown).
T cells play a central role in the generation of GVHD after ABMT. Although T cells are mediators of immunity, their function is under the control of APCs. Therefore, APCs may regulate the activation of host-specific donor T cells, which are responsible for the generation of GVHD. Our present results show that blockade of the B7/CD28 pathway in anergizing priming MLR cultures leads to a distinct program of activation and differentiation of monocytes into macrophages, which acquire distinct gene expression and functional properties as compared with macrophages generated in MLR cultures in medium alone. This altered macrophage differentiation program closely resembles what has been defined as "alternative activation of antigen-presenting cells."13,14 Macrophages isolated from anergizing cultures have increased expression of CD14, MHC class II, CD23, and scavenger receptors and increased phagocytotic activity, but reduced ability to process and present antigen. Moreover, these macrophages mediate immunosuppressive effects to T cells. In our recent clinical trial that allowed successful haploidentical
BMT, recipient PBMCs were collected, irradiated, and cocultured with
donor bone marrow ex vivo in the presence of CTLA4-Ig to block B7-1-
and B7-2-mediated costimulation, washed, and infused into the
patient.8 All treated patients rapidly achieved
engraftment and developed full donor chimerism and no clinically
detectable GVHD. Although in vitro analysis during that study indicated
that anergy had been induced to host-specific donor T cells, our
present results indicate that additional mechanisms may be responsible for the clinical outcome of this treatment. As we show here, anergizing cultures result in the generation of AAM The cytokine profile of the anergic cells is characterized by defective
production of IFN- An additional mechanism responsible for the generation of AAM Several studies have shown an important role of IL-10 in the regulation
of the differentiation and activation program of
APCs.13,32,38 IL-10 prevents the differentiation of
monocytes to dendritic cells and promotes their maturation into
macrophages.32 These macrophages that develop in the
presence of IL-10 have increased endocytotic activity but defective
ability for antigen presentation. Although IL-10 blocks the
differentiation of CD14+ monocytes to dendritic cells
induced by granulocyte macrophage colony-stimulating factor and IL-13,
the macrophages that develop under these conditions have cytochemical
and phenotypic features of mature macrophages. Thus, the presence of
IL-10 does not simply prevent activation of monocytes, but initiates an
activation program that preferentially leads to their differentiation
into mature macrophages instead of dendritic cells. Our present studies
show that the defective ability of macrophages differentiated in the presence of IL-10 to induce T-cell activation is linked to their defective ability to process and present antigen. At the same time,
AAM Consistent with these functional findings, biochemical evidence from another study supports the notion that IL-10 does not simply deactivate monocytes, but leads to activation of selective signaling pathways.55 IL-10 blocks LPS-mediated activation of p56lyn, phosphorylation of vav, and activation of Ras in monocytes, but does not inhibit p56lyn-mediated induction of c-jun and c-fos.55 Thus, selective predominance of signaling pathways during monocyte activation in the presence of IL-10 results in distinct gene transcription and distinct functional properties. These developing observations about the significance of the distinct differentiation programs of APCs in the regulation of the immune response13,48 may have important implications in the field of transplantation. Clinical data and results from in vivo animal models have shown that allografts of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs) have an unexpected immunologic behavior.56,57 Transplantation of allogeneic G-CSF-mobilized PBSCs, which contain 10-fold higher numbers of T cells as compared with bone marrow grafts, did not result in a higher incidence of GVHD.58,59 Moreover, such grafts achieved better engraftment across the HLA barrier.60,61 Potentially, CD14+ cells contained in the G-CSF-mobilized PBMCs have an active role in the reduced responsiveness of the CD4+ cells contained in this population.62 These CD14+ cells are involved in the impaired transactivation of the CD28 response element (CD28RE) of G-CSF-mobilized CD4+ cells.63 It was shown that such CD14+ cells produce high levels of IL-10 and mediate their effects in an IL-10-dependent manner.64 Studies in animal models have indicated that T cells from
G-CSF-treated animals preferentially produce IL-4 and IL-10 and are
associated with diminished ability to induce GVHD.57,65 Consistent with these experimental data, G-CSF-mobilized PBSCs from
healthy donors contain high numbers of type-2 dendritic cells (DC2),
which prime T cells to produce IL-4 and IL-10.66
Interestingly, umbilical cord blood, another source of allogeneic stem
cells for transplantation associated with a low incidence of acute
GVHD, contains DC2 and not DC1-type cells.67 These studies
suggest that donor APCs regulate both GVHD induction and engraftment
and have a critical role in the clinical outcome of allogeneic
transplantation. Consistent with these observations, in our system
IL-10 was produced by CD4+ and not by CD14+
cells. Moreover, our experiments showed a reciprocal regulation between
T cells and macrophages. Anergic T cells generated during blockade of
B7/CD28 interaction produce high amounts of IL-10, which mediates
generation of AAM An additional interesting finding of our study with potential clinical
significance is the observation that blockade of B7/CD28 interactions
leads to the generation of AAM In conclusion, our results show that induction of T-cell anergy by
blockade of B7/CD28 costimulation influences the maturation and
differentiation process of macrophages, which subsequently have an
inhibitory role on T-cell responses. This novel and unexpected observation that anergic T cells mediate the generation of AAM
Submitted August 7, 2001; accepted October 11, 2001.
Supported by National Institutes of Health grants AI 43552, AI 41584, and HL 54785.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Vassiliki A. Boussiotis, Dana-Farber Cancer Institute, Mayer 547, 44 Binney St, Boston, MA 02115; e-mail: vassiliki_boussiotis{at}dfci.harvard.edu. |
Top
Abstract
Introduction
RII) and major
histocompatibility complex (MHC) class II. They have enhanced capacity
for phagocytosis and scavenging, but decreased capacity for nitric
oxide (NO) and O2 production and antigen
processing.
(MIP-1
