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* Dumont-University of California Transplant Center, University of California School of Medicine, Los Angeles, CA 90095;
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205; and
Laboratory of Immunogenetics and Transplantation, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD154 (CD40 ligand), a member of the TNF gene family, is expressed predominantly on mature activated CD4+ T cells and other cell types, but not on resting T cells (4). The interaction between CD154 and CD40, a glycoprotein receptor on APCs, provides a costimulatory signal that is essential for development of both cellular and humoral immune responses to T-dependent Ags (5, 6). The efficacy of CD154-targeted therapy to abrogate the rejection response and to markedly prolong allograft survival in rodents and subhuman primates has been well established (7, 8, 9). This highlights the role of the CD154-CD40 activation pathway in the immune cascade leading to acute allograft rejection. However, the actual mechanism by which CD154 blockade interferes with host alloimmune responses remains to be elucidated. Recently it has been shown that, although anti-CD154 mAb treatment prevented CD4+ T cell-mediated rejection, it may have little or no effect on CD8+ T cell activation, proliferation, differentiation, and homing to the target organ (10). Apart from a single report in which CD154 mAb treatment ameliorated blood transfusion-induced sensitization to pancreatic islets (11), little is known about the role of CD154 signaling in sensitized hosts. It also remains to be determined whether the blockade of CD154 costimulatory signals in sensitized hosts affects graft rejection via CD4- or CD8-dependent mechanism.
To investigate mechanisms of host sensitization to alloantigens in
vivo, and to further address the role of CD154 in the rejection
cascade, we have developed a stringent model of vascularized cardiac
allograft rejection in skin-sensitized mice (BALB/c
B6/129;
accelerated rejection in <36 h) (12). The sequence of
events in this experimental system is relevant to clinical cases of
rejection in patients sensitized to MHC Ags before transplantation. In
this murine model, the evidence of cellular immune activation includes
generation of high CTL activity in vitro and infiltration of the graft
by mononuclear cells expressing IL-2, IFN-
, TNF, and IL-2R. The
presence of antidonor Abs and extensive intragraft deposition of IgM,
IgG, C3, and rapid neutrophil infiltration confirm cardinal features of
humoral injury.
This study analyzes the mechanisms by which CD154-CD40 costimulation may affect host sensitization to alloantigen in vivo. First, we determined that CD8+, but not CD4+, T cells are the prime effectors in executing accelerated rejection. Then, by using CD154-/- mice as recipients of skin and cardiac allografts, and by selectively targeting CD154-CD40 in vivo interactions in wild-type (WT)4 hosts, we confirmed the key role of the CD154 pathway in sensitized hosts. Indeed, although cardiac allografts were rejected in <36 h in WT skin-sensitized mice, they survived >100 days in CD154-deficient mice or WT mice treated with anti-CD154 mAb. In contrast, CD40-deficient mice with disrupted CD154-CD40 interactions selectively in the indirect allorecognition (for CD8+ T cells) had a normal kinetics of accelerated rejection. Thus, the CD154 blockade in the direct allorecognition pathway is critical in host allosensitization. The results of our studies are the first to document that CD154 blockade in sensitized hosts results in selective inhibition of alloreactive CD8+ but sparing of CD4+ T cell responses, both systemically and locally.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WT BALB/c (B/c; H-2d), B6/129 (B6;
H-2b), and CBA/Ca (CBA;
H-2k) male mice were used. We also used
CD154-/- and CD40-/-
mice of the B6 background (intercrossed at least 10 generations). All
mice (8–12 wk old; 20–25 g) were obtained from The Jackson Laboratory
(Bar Harbor, ME) and were housed in the University of California animal
facilities (Los Angeles, CA) under pathogen-free conditions. Orthotopic
full-thickness skin grafts (
0.5 cm in diameter) from B/c donors were
sutured bilaterally onto the flanks of prospective WT,
CD154-/-, or CD40-/- B6
recipients. These were then challenged 10 days later into the
intra-abdominal location with vascularly anastomosed B/c cardiac
grafts. Graft survival was assessed daily by palpation of ventricular
activity. The day of rejection was defined as the day of cessation of
heartbeat and was verified by autopsy and pathological examination.
Ab therapy
Rat anti-mouse CD4 (GK1.5)- and CD8 (2.43; courtesy of Dr. H. Auchincloss, Massachusetts General Hospital, Boston, MA)-depleting mAb were administered at days -2, -1, and 0 in skin-sensitized WT recipients (0.2–0.4 mg per mouse i.v.). Anti-CD154 Ab (MR1), purchased from Bioexpress (West Lebanon, ME), was given in the sensitization phase, i.e., between skin and cardiac engraftment (0.25–0.5 mg per mouse every other day i.v.). Control recipients were treated with relevant doses of rat or hamster Ig.
In vitro MLR
Spleens and peripheral lymph nodes were removed and passed through a cytoscreen into RPMI 1640 (Life Technologies, Grand Island, NY). The cells and residue were pelleted at 1200 rpm for 5 min and then resuspended in 5 ml Tris-ammonium chloride buffer (0.83% NH4Cl, 5 mM Tris buffer (pH. 7.2)) at 37°C for 5 min to lyse RBC. After washing twice with RPMI 1640 medium with 1% FBS, cells were suspended in culture medium (RPMI 1640 supplemented with 20 mM HEPES, 10 mM sodium pyruvate, 2 mM L-glutamine, 50 nM 2-ME, 1x MEM-nonessential amino acid solution, 1x MEM-vitamin solution, 1x antibiotic/antimycotic solution, and 10% FBS) at a concentration of 5 x 106/ml. A total of 100 µl of responder B6 cells were added into a U-bottom 96-well plate (Corning, Corning, NY) and were mixed with the same number of x-irradiated (2000 rad) stimulator cells of B6 (syngeneic), B/c (donor-type), or CBA (third-party) strains. Four replicates were used for each reaction combination. Con A (2 mg/ml) was used as a positive control. A total of 1 µCi of [3H]TdR was added to each well in the last 16–18 h of a 3-day culture. Labeled cells were harvested onto filter mats (Skatron Instrument, Sterling, VA) with a Skatron 12-well cell harvester. The cpm of the filter membranes were measured in scintillation liquid on a Beckman LS 6000IC (Beckman Coulter, Fullerton, CA).
In vivo MLR
In in vivo MLR, responder cells (B6 strain) were labeled with CFSE (Molecular Probes, Eugene, OR) at 4 mM in PBS for 15 min at 37°C. The unconjugated CFSE was eliminated by washing the cells with FBS (20%) supplemented RPMI 1640 medium. The labeled cells were resuspended in PBS at 2.5 x 108/ml, and 200 µl of these cells (5 x 107) were injected into total-body x-irradiated (1000 rad) B/c (or B6 as negative controls) mice via tail vein. At day 3, spleens of the injected mice were harvested and stained with anti-mouse CD3e-R-PE (clone 145-2C11), CD4-biotin (clone H129.19), and streptavidin-CyChrome (BD PharMingen, San Diego, CA). Topro 3 (1 nM) was added as viable dye. Four-color flow cytometry was performed on a FACSCalibur dual-laser cytometer (BD Biosciences, Mountain View, CA). Topro 3-negative cells in lymphocyte gate (viable cells) that stained positive for CD3 and CD4 were analyzed for CFSE intensities.
CTL effector differentiation in vivo
RBC-free splenocytes or lymph node cells were prepared as described above. One million cells were used for Ab staining in ice-cold PBSA (PBS with 1% BSA). Cells were first incubated with 10 µg of normal rat IgG to block Fc binding sites. After washing, cells were stained with 0.5–1 µg of rat anti-mouse CD8a-FITC (clone 53-6.7), CD62L-R-PE (clone MEL-14), and CD44-CyChrome (clone IM7) (BD PharMingen). After washing, three-color flow cytometry was performed on a FACScan cytometer (BD Biosciences). Cells in lymphocyte gate that stained positive for CD8a were analyzed for their CD62L and CD44 expression. CTL effectors were identified as the CD8+CD62LlowCD44high population.
In vitro CTL assay
B6 mouse splenocytes were obtained and cultured in bulk against gamma-irradiated donor B/c splenocytes for 6 days. Viable lymphocytes were counted and set up against 51Cr-labled B/c target cells (Con A blast from 3- to 4-day cultures) in a U-bottom 96-well plate at different ratios. After a 6-h incubation, supernatants were harvested and measured for gamma activity. Supernatants from wells of the target cells alone were counted as spontaneous release, and those from wells of target cell with 25% Triton X-100 as maximal release. The specific cytolysis was calculated as follows: % = (cpmsamples - cpmspontanous)/(cpmmax - cpmspontanous).
Histology and immunohistology
H&E and immunoperoxidase staining were used for assessment of myocardial and vascular preservation and detection of inflammatory cell infiltration and IgM/IgG deposition, as described (14). Tissues were fixed in buffered formalin or embedded in Tissue Tec OCT compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -70°C. Cryostat sections (5 µm) were fixed in acetone and endogenous peroxidase activity was blocked by incubating the slides with 0.3% H2O2. Normal heat-inactivated rabbit serum was used for blocking. Appropriate primary rat Ab against mouse CD3+ cells (CD3-12), CD4+ cells (CT-CD4), CD8+ cells (2.43), and monocytes/macrophages (MOMA-2) were used (Harlan Bioproducts for Science, Indianapolis, IN). Primary Abs against mouse IgG and IgM, biotinylated rabbit anti-rat IgG, and streptavidin peroxidase-conjugated complexes were obtained from Vector Laboratories (Carpinteria, CA). The control sections were performed by replacing the primary mAb with either dilution buffer or normal rat serum. The peroxidase reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO). The sections were evaluated blindly by counting labeled cells within 10 high-power fields per section. Some Ags were analyzed in a semiquantitative fashion where the relative abundance of each one was judged as follows: negative (-), little (+), moderately abundant (++), and very abundant (+++).
Alloantibody levels
Antidonor Abs were measured by flow cytometry. Briefly, 50-µl aliquots containing 1.5 x 105 B/c lymph node lymphocytes were incubated for 45 min at 4°C with 50 µl of diluted sera (1/4, 1/16, 1/64). To stain for IgM and IgG, cells were washed twice and then reacted with 50 µl of PBS plus 0.2% BSA plus 0.02% sodium azide containing a mixture of FITC-conjugated goat Ab specific for the Fc portion of mouse IgG and PE-conjugated goat Ab specific for the µ-chain of mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA). After staining for 30 min at 4°C, the cells were washed, fixed in 1% formaldehyde in PBS, and analyzed by flow cytometry using FACScan (BD Biosciences). The binding of IgM and IgG alloantibodies to class I MHC Ags on B/c target cells is expressed as the mode channel fluorescence.
Statistical analysis
Comparisons were made using the unpaired two-tailed Student’s t test. Differences in values at p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/c hearts were rejected in 1.4 ± 0.3 days (mean survival
time (MST) ± SD) in B6 WT mice that were sensitized with B/c skin
grafts at day -10 (Fig. 1
). Two groups
of WT recipients of skin grafts underwent a 3-day treatment with
depleting anti-CD4 or anti-CD8 mAb (day -2 to day 0). Such a
delayed (8 days post-skin engraftment) depletion of T cell subsets
should not disrupt T cell activation by skin grafts (sensitization),
yet it should prevent alternative activation pathways leading to
accelerated graft rejection. Flow cytometry confirmed complete
depletion of the targeted T cell subset while maintaining both the
number and function of the opposite subset (data not shown).
Interestingly, as shown in Fig. 1A, unlike animals treated
with anti-CD4 mAb, which lost their cardiac allografts uniformly
within 24 h, those given anti-CD8 mAb rejected their
transplants in an acute rather than accelerated manner (5.5 ± 1
days; p < 0.001). Thus, activated
CD8+ but not CD4+ T cells
are the principle effectors in executing accelerated rejection of
cardiac allografts in this sensitized mouse transplant model.
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Cardiac allografts survive long-term after disruption of CD154 costimulation in sensitized mice
A group of CD154-/- B6 mice was then
challenged with B/c skin, followed 10 days later by transplantation of
B/c hearts. As shown in Fig. 1
B, all cardiac allografts
survived >100 days in CD154-/- recipients. In
contrast, skin grafts alone (no heart grafts) were rejected in 12
± 2 days, whereas heart grafts alone (no skin grafts) survived >100
days in otherwise unmodified CD154-/- hosts
(n = 4 mice per group; data not shown). To demonstrate
that these findings were not confined to the murine knockout system, in
which the lack of CD154 expression is complete, sustained, and
specific, we then used anti-CD154 mAb treatment to block CD154-CD40
in vivo interactions in WT mice. Indeed, infusion of MR1 mAb every
other day between skin (day -10) and cardiac (day 0) engraftment
prevented host sensitization, as evidenced by long-term cardiac
allograft acceptance in 8 of 10 WT hosts (Fig. 1B). In
contrast, controls conditioned with hamster serum rejected cardiac
allografts in <2 days.
Prevention of host sensitization by disruption of CD154-CD40 signaling results from inhibition of direct T cell activation by donor APCs
Both direct and indirect T cell activation can be involved in host
sensitization to alloantigen. To determine which activation pathway is
dominant in our model, we took advantage of the finding that
CD8+ T cells were the main effectors in our model
and that CD40-deficient (B/6) recipients were defective only in the
indirect pathway for alloreactive CD8+ T cell
activation, as allogeneic APCs in WT B/c grafts have normal CD40
expression. This is in contrast to CD154-deficient recipients,
defective in both direct and indirect pathways, due to the lack of
CD154 expression on responding T cells. A group of
CD40-/- (B6) animals was challenged with skin
grafts (B/c), followed 10 days later by transplantation of donor-type
hearts. Unlike CD154-/- skin-sensitized mice,
which maintained their transplants long-term, all cardiac grafts were
rejected promptly in CD40-/- recipients
(1.5 ± 0.5 days; Fig. 1C), comparable with
skin-sensitized WT hosts. Thus, CD154-CD40 signaling that is involved
in the direct CD8+ T cell activation pathway by
donor APCs is critical in host sensitization to alloantigen in
vivo.
T cell hyporesponsiveness to alloantigen in vitro is restricted to the CD8+ subset from CD154-/- recipients
Spleen lymphocytes were harvested from groups of WT and
CD154-/- primed recipients of cardiac
allografts at 1–2 days posttransplant and analyzed for in vitro
alloreactivity in MLR assay. By that time, WT hosts undergo fulminant
rejection of cardiac allografts, whereas CD154-deficient mice maintain
functioning transplants despite prior donor-type skin engraftment. As
shown in Fig. 2A, splenocytes
from CD154-deficient hosts proliferated less vigorously against
alloantigens, as compared with WT controls (donor-type,
p < 0.0005; third-party, p < 0.05).
Addition of Con A stimulated proliferation of T cells to similar
degrees in both recipient groups, indicating no general proliferation
defects in splenocytes from CD154-/-
recipients. FACS staining of CFSE-labeled splenocytes in bulk cultures
identified both CD4 and CD8 phenotypes in the alloresponsive T cell
population. Whereas CD154-/-
CD4+ T cells proliferated normally as compared
with WT CD4+ T cells (16.6 vs 9.1%, Fig. 2B), the CD154-/-
CD8+ T cells had much-reduced numbers of
proliferating cells compared with the WT CD8+ T
cells (24.8 vs 70.1%, Fig. 2B). These results indicate in
vitro hyporesponsiveness of CD154-/-
splenocytes after in vivo priming, and that this hyporesponsiveness was
restricted to CD8+ T cells without affecting the
CD4+ T cell compartment.
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To further examine the proliferative response of
CD4+ T cells, we conducted in vivo MLRs, which
provide an ideal setting for alloreactive CD4+ T
cells to differentiate between deletional and nondeletional mechanisms
that may contribute to hyporesponsiveness in vitro. Hence, spleen cells
from groups of WT and CD154-deficient primed hosts were harvested at
days 1–2 posttransplant, labeled with CFSE, and then injected i.v.
into gamma-irradiated donor-type (B/c) test mice to determine their in
vivo alloreactivity. The results presented in Fig. 3 are in agreement with robust
proliferation of transferred spleen cells from
CD154-/- hosts, similar to that of rejecting WT
littermates. CD4+ T cells were the major
responsive population. This response was allospecific, as no
significant proliferation was detected with cells injected into
gamma-irradiated syngeneic (B6) mice. These results confirm our in
vitro results that alloreactive CD4+ T cells were
responsive in CD154-deficient hosts.
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To further examine the function of alloreactive
CD8+ cells from CD154-/-
recipients, splenocytes were analyzed for the generation of
CD8+ CTL effector phenotype induced by
alloantigens. The expression of the
CD8+CD44highCD62Llow
phenotype correlates with cytolytic activity, as previously shown
(13). Fig. 4A
illustrates results of a representative experiment and depicts the
percentages of
CD8+CD44highCD62Llow
spleen cells in WT and CD154-/- engrafted hosts
at day 2 posttransplant. Unlike in primed WT splenocytes, the CTL
effector phenotype expression by CD154-deficient splenocytes was
markedly diminished, and was comparable with that of naive mice (43.1,
4.7, and 2.3%, respectively). These data were consistent with results
of an in vitro cytotoxicity assay (Fig. 4B), in which target
cell killing by CD154-/- T cells was markedly
diminished as compared with that of WT cells (12 ± 3 and 53
± 4%, respectively, at a 50:1 E:T ratio). Collectively, these results
document abrogation of CD8+ T cell
differentiation and generation of
CD8+CD44highCD62Llow
phenotype CTL effector cells, consistent with defective effector T cell
function in mice deficient for CD154.
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We then asked how disruption of the CD154 costimulation pathway
affects host cell immune responses at the graft site. The sequential
analysis of cardiac allografts harvested from sensitized WT mice at
24–30 h posttransplant showed considerable edema and myocyte
degeneration, contrasting with the relatively normal histological
appearance observed in the cardiac grafts harvested from primed
CD154-/- hosts. Fig. 5 depicts some of the key
immunohistological findings in cardiac allografts from WT and
CD154-/- recipients. Thus, cardiac grafts from
WT hosts were characterized by elevated myocardial infiltration,
consisting predominantly of CD3+ cells (Fig. 5A; 40.5 ± 4.9). In contrast, cardiac grafts from
CD154-deficient hosts showed depression in the number of
CD3+ cells (Fig. 5G; 17.2 ± 8.8;
p < 0.02). Interestingly, unlike
CD4+ cells, which remained comparable between WT
(Fig. 5B; 7.5 ± 0.7) and CD154-deficient (Fig. 5H; 6.3 ± 2.1) mice, the frequency of infiltrating
CD8+ cells diminished sharply in
CD154-/- hosts (Fig. 5I; 26 ±
8.7) as compared with WT controls (Fig. 5C; 71.5 ±
16.2; p < 0.01). A similar cell infiltration pattern
was detected in cardiac allografts harvested at 24–30 h from WT
skin-sensitized mice treated with MR1 mAb (data not shown). Hence,
consistent with our findings on cells in the peripheral lymphoid
organs, CD154 signals are also required for the migration of
CD8+ T cells to the alloantigen site, as their
absence (in CD154-/- hosts) or blockade (in MR1
mAb-treated WT mice) prevented sequestration of
CD8+ T cells in cardiac allografts in this
model.
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Because the CD154-CD40 pathway is critical for CD4-dependent B
cell reactions, we determined systemic donor-specific alloantibody
responses elicited by B/c cardiac allografts in B6 mice sensitized with
B/c skin grafts. Sera were harvested from groups of untreated WT and
CD154-deficient recipients at days -10 (the day of skin graft), -7,
-3, 0 (the day of cardiac engraftment), +1, +2, and +7. Serially
diluted sera samples were then tested by flow cytometry for IgM and IgG
binding to B/c lymph node target cells. In sensitized WT engrafted
hosts, IgM alloantibody response peaked at day +2, i.e., around the
time of accelerated rejection (Fig. 6).
The IgM alloantibody response switched to strong IgG alloantibody
response, which remained elevated for up to 7 posttransplant days. In
contrast, in CD154-/- recipients, serum IgM and
IgG alloantibody levels remained close to background levels throughout
the observation period.
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, cardiac allografts
from WT mice showed extensive and widespread deposits of IgM (Fig. 5E; ++) and IgG (Fig. 5F; +++). In contrast,
cardiac grafts from CD154-deficient hosts were characterized by
diminished staining for IgM (Fig. 5K; +) and IgG (Fig. 5L; +/-). Thus, disruption of the CD154 costimulation
pathway abrogates otherwise strong systemic and intragraft antidonor
IgM and IgG alloantibody responses in primed recipients. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously reported, the interaction between CD40 on B cells and CD154 on T cells plays a key role in B cell activation, including proliferation, the generation of a thymus-dependent Ab response, and Ig isotype switching (17). Indeed, treatment with anti-CD154 mAb diminished the response to alloantigen on B cells in vitro (18), whereas immunization with resting B cells from CD154-/- mice induced tolerance to alloantigen (19). Treatment with anti-CD154 mAb blocks both primary and secondary immune responses to T cell-dependent Ags, as well as the development of germinal centers and the generation of memory cells (20). Moreover, individuals with type IgM syndrome, an immunodeficiency due to a mutation of the CD154 gene, express diminished serum IgG, IgA, and IgE levels and are unable to mount secondary T cell-dependent responses (21). Similarly, we found diminished IgM and IgG alloantibody levels in the serum of CD154-deficient recipients of both skin and heart allografts. Moreover, intragraft deposition of both IgM and IgG was depressed in well-functioning grafts of CD154-/- hosts as compared with that in rejecting WT controls. All these suggest that depression of Ig responses and class switching represents a relevant mechanism by which disruption of the CD154 costimulation pathway prevents sensitization to alloantigen in vivo.
As to the T cell responses, the results of our study demonstrate that
splenic T cells from CD154-deficient hosts were hyporesponsive to
alloantigen in in vitro MLR, as compared with WT controls, consistent
with a previous report by Shimizu et al. (22). However,
subset analysis of the proliferating cells has revealed that the
overall hyporesponsiveness in CD154-deficient hosts resulted from
selective depression of CD154-deficient CD8+ T
cells, whereas proliferation of CD154-deficient
CD4+ T cells was virtually unaffected. In
addition, CFSE-labeled CD4+ splenocytes from
CD154-/- recipients proliferated vigorously in
vivo following infusion into x-irradiated allogeneic test mice, similar
to cells from WT controls. This indicates that alloreactive
CD4+ T cells in the splenocyte pool were
unaffected in our CD154-/- transplant model, at
least in their proliferative responses and cytokine production
(IFN-; data not shown) against alloantigens. In contrast,
CD8+ T cells were both hyporesponsive in vitro
and defective in their ability to become activated in vivo
(differentiation into CTL effectors) by allograft stimulation. This
defect was alloantigen specific, as polyclonal stimulation, such as
with Con A or PMA/ionomycin, readily stimulated
CD154-/-CD8+ T cells to
proliferate and secrete cytokines (IFN-; data not shown) to the same
extent as WT CD8+ T cells. To best of our
knowledge, this is the first direct evidence that in an allograft model
alloreactive CD8+ but not
CD4+ T cells were severely hampered by the CD154
blockade. It is worth pointing out that, although CD154-deficient
CD4+ T cells were able to proliferate, their
functions may still be interrupted, particularly in the aspect of
providing help for CD8+ T cells and B cells.
The mechanism by which disruption of the CD154-CD40 pathway suppresses cell-mediated immunity varies depending on the experimental model system. Although it is well established in nominal Ag model systems that CD4-dependent CD8+ T cell activation requires CD154-CD40 costimulation via indirectly activated dendritic cells by CD154-positive CD4+ T cells (23, 24), its role in alloreactive CD8+ T cells remains controversial. Alloreactive CD8+ T cells are present in high precursor frequencies and may become activated through both CD4-dependent and CD4-independent pathways. Unlike CD4-dependent CD8 activation, the requirement for CD154 costimulation in CD4-independent CD8+ T cell activation remains largely unknown. The existing literature favors the hypothesis that CD8+ alloreactive T cells, as a whole population, are less sensitive or resistant to the CD154 blockade, as shown by Jones (10), Guo (25), and Honey (26) in both major and minor MHC-mismatched allograft models. However, reports from allogeneic tumor models suggest that CD8+ T cell activation may depend on the CD154-CD40 pathway, and that activation of CD8+ T cells may be in fact CD4 independent (15, 16). In addition, it has been shown that long-term expansion of adoptively transferred 2C TCR-transgenic CD8+ T cells in Ag-bearing F1 hosts depends on CD154 costimulation signals via CD4+ T cells (27). Our results document that disruption of CD154 signaling was indeed inhibitory to alloreactive CD8+ T cells in sensitized transplant recipients, and our preliminary data suggest that both CD4-dependent and CD4-independent activation of alloreactive CD8+ T cells may be affected in this model (Y. Zhai, manuscript in preparation).
The sparing of at least some of alloreactive CD4+ T cell function (proliferation and cytokine production) by CD154 blockade is of interest. In contrast to cells lacking CD28 signaling, which were unable to proliferate in both CD4 and CD8 compartments in vitro and in vivo (data not shown), CD154-deficient CD4+ T cells or WT CD4+ T cells under MR1 treatment all underwent vigorous proliferation after allostimulation. This may be important for long-term cardiac allograft survival after transient CD154 blockade in our model, because CD28-deficient recipients promptly lost their transplants in <2 days, whereas treatment with CTLA4-Ig only marginally prolonged cardiac allograft survival in skin-sensitized WT mice (data not shown). A similar observation (effects of CD154 vs CD28 blockade) was reported recently in a murine obstructive airway disease model (28). The beneficial effects of CD4 activation may relate to the activation of regulatory CD4+ T cells, critical for long-term graft survival and tolerance, particularly after transient immunosuppressive protocols. In the absence of immunosuppression, alloreactive lymphocytes do recover or generate de novo. Regulatory T cells are the main force at this stage to control these aggressive cells.
In conclusion, by demonstrating selective inactivation of alloreactive CD8+ T cells as the consequence of disrupting the CD154-CD40 costimulation pathway that prevents accelerated rejection, this study provides new insights into divergent costimulatory requirements for T cell subsets. Our data are consistent with the key role of CD154 signaling in the mechanism of host sensitization to alloantigen.
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Acknowledgments |
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Footnotes |
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2 M.H.S. and J.W.K.-W. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jerzy W. Kupiec-Weglinski, Dumont-University of California Transplant Center, Room 77-120 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: jkupiec{at}mednet.ucla.edu
4 Abbreviations used in this paper: WT, wild type; MST, mean survival time.
Received for publication March 29, 2002. Accepted for publication May 16, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during the differentiation of cytotoxic T lymphocytes induced by allografts. J. Immunol. 148:2348.[Abstract]
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Y. Zhai, L. Meng, F. Gao, Y. Wang, R. W. Busuttil, and J. W. Kupiec-Weglinski CD4+ T Regulatory Cell Induction and Function in Transplant Recipients after CD154 Blockade Is TLR4 Independent J. Immunol., May 15, 2006; 176(10): 5988 - 5994. [Abstract] [Full Text] [PDF]
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K. E. Lunsford, M. A. Koester, A. M. Eiring, P. H. Horne, D. Gao, and G. L. Bumgardner Targeting LFA-1 and CD154 Suppresses the In Vivo Activation and Development of Cytolytic (CD4-Independent) CD8+ T Cells J. Immunol., December 15, 2005; 175(12): 7855 - 7866. [Abstract] [Full Text] [PDF]
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T. Ito, T. Ueno, M. R. Clarkson, X. Yuan, M. M. Jurewicz, H. Yagita, M. Azuma, A. H. Sharpe, H. Auchincloss Jr, M. H. Sayegh, et al. Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo J. Immunol., June 1, 2005; 174(11): 6648 - 6656. [Abstract] [Full Text] [PDF]
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 J. L. Ferrant, C. D. Benjamin, A. H. Cutler, S. L. Kalled, Y.-M. Hsu, E. A. Garber, D. M. Hess, R. I. Shapiro, N. S. Kenyon, D. M. Harlan, et al. The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge Int. Immunol., November 1, 2004; 16(11): 1583 - 1594. [Abstract] [Full Text] [PDF]
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L. Quemeneur, L. Beloeil, M.-C. Michallet, G. Angelov, M. Tomkowiak, J.-P. Revillard, and J. Marvel Restriction of De Novo Nucleotide Biosynthesis Interferes with Clonal Expansion and Differentiation into Effector and Memory CD8 T Cells J. Immunol., October 15, 2004; 173(8): 4945 - 4952. [Abstract] [Full Text] [PDF]
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L. Nagelkerken, I. Haspels, W. van Rijs, B. Blauw, J. L. Ferrant, D. M. Hess, E. A. Garber, F. R. Taylor, and L. C. Burkly FcR Interactions Do Not Play a Major Role in Inhibition of Experimental Autoimmune Encephalomyelitis by Anti-CD154 Monoclonal Antibodies J. Immunol., July 15, 2004; 173(2): 993 - 999. [Abstract] [Full Text] [PDF]
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 T. El-Sawy, M. Miura, and R. Fairchild Early T Cell Response to Allografts Occuring Prior to Alloantigen Priming Up-Regulates Innate-Mediated Inflammation and Graft Necrosis Am. J. Pathol., July 1, 2004; 165(1): 147 - 157. [Abstract] [Full Text] [PDF]
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R. Abdi, T. K. Means, T. Ito, R. N. Smith, N. Najafian, M. Jurewicz, V. Tchipachvili, I. Charo, H. Auchincloss Jr., M. H. Sayegh, et al. Differential Role of CCR2 in Islet and Heart Allograft Rejection: Tissue Specificity of Chemokine/Chemokine Receptor Function In Vivo J. Immunol., January 15, 2004; 172(2): 767 - 775. [Abstract] [Full Text] [PDF]
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Y. Zhai, L. Meng, R. W. Busuttil, M. H. Sayegh, and J. W. Kupiec-Weglinski Activation of Alloreactive CD8+ T Cells Operates Via CD4-Dependent and CD4-Independent Mechanisms and Is CD154 Blockade Sensitive J. Immunol., March 15, 2003; 170(6): 3024 - 3028. [Abstract] [Full Text] [PDF]
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Y. Zhai, L. Meng, F. Gao, R. W. Busuttil, and J. W. Kupiec-Weglinski Allograft Rejection by Primed/Memory CD8+ T Cells Is CD154 Blockade Resistant: Therapeutic Implications for Sensitized Transplant Recipients J. Immunol., October 15, 2002; 169(8): 4667 - 4673. [Abstract] [Full Text] [PDF]
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, sensitized mice treated with anti-CD4 mAb (MST = 2.0
days; n = 4); 
, sensitized mice treated
with anti-CD8 mAb (MST = 5.0 days; n = 4);
, CD154-/- mice (MST = >100 days;
n = 9); 
, WT mice treated with anti-CD154
(MR1) mAb (every second day between days -10 and -2; MST = >100
days; n = 10); 
, WT mice treated with control
hamster Ig (MST = 1.9 days; n = 4).
C, The survival of B/c cardiac allografts in B6 mice
sensitized with B/c skin grafts (day -10). 
