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Departments of
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Medicine and
Pathology, 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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50% in CD40L-/- hosts, the relative
percentages of macrophages and T cell subsets were comparable to WT.
IFN-
, TNF-
, and IL-10 were diminished commensurate with the
reduced cellular infiltrate; IL-4 was not detected.
CD40L-/- recipients did not develop IgG alloantibodies
and showed diminished B7 and CD28 expression on subsets of
graft-infiltrating cells. CD40L-/- transplant recipients
developed allospecific tolerance to the donor haplotype; second set
donor skin grafts engrafted well, whereas third-party skin grafts were
vigorously rejected. By MLR, splenocytes from CD40L-/-
allograft recipients also demonstrated allo-specific
hyporesponsiveness. Nevertheless, allografts in CD40L-/-
hosts developed significant graft arteriosclerosis by 8–12 wk
posttransplant. Therefore, we propose that early alloresponses, without
CD40-CD40L costimulation, induce allospecific tolerance but may trigger
allo-independent mechanisms that ultimately result in graft
vasculopathy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40, a 50-kDa glycoprotein member of the TNF receptor family, is expressed on B cells, dendritic cells, basal epithelial cells, macrophages, Langerhans cells, endothelial cells, and thymic epithelial cells (5). CD40L (CD154), a type II integral membrane glycoprotein and a member of the TNF family, engages and stimulates CD40. Pharmacological activation of CD4+ T cells or stimulation of the TCR with mAbs induces transient expression of CD40L (6).
Using pigeon cytochrome c-specific TCR transgenic mice, Roy et al. (6) showed that TCR interactions with MHC/Ag complexes on APCs together with leukocyte function-associated Ag-1, induced expression of CD40L on the responding T cells. In turn, CD40L interaction with CD40 on APCs induced increased expression of B7-1 (CD80) and B7-2 (CD86). Therefore, CD40L is critical for B cells and other APCs to express high levels of costimulatory molecules, and for these cells to become competent "professional" APCs.
Recent work has established the importance of costimulation in the development and maintenance of immunity (4, 7). Blockade of the CD40-CD40L pathway therefore seems an attractive strategy to manipulate the T cell-dependent immune response, including allograft rejection. In experimental models, Ab blockade of CD40-CD40L interactions inhibits graft-vs-host disease and the generation of cytotoxic T cell responses, as well as markedly prolonging allograft survival. Although these effects on T cell activation may be related to functional dysregulation of B7-CD28 costimulation, the mechanisms of long-term survival remain unclear. Given the broad distribution of CD40 and CD40L (5, 8), it is also uncertain whether the effects of mAb are due to effects on graft cells or on host immune responses. Finally, the reported effects of CD40-CD40L blockade on graft arterial disease (GAD), a major impediment to long-term human allograft survival, are conflicting. Hancock et al. (9) showed that a transient blockade of CD40-CD40L pathway using mAb directed against the ligand for CD40 (MR-1) combined with donor cell infusion enhanced allograft survival and prevented GAD in a mouse heart transplant model. However, Larsen et al. (10) demonstrated that anti-CD40L mAb treatment induced long-term allograft survival but had minimal beneficial effect on posttransplant arteriopathy. Moreover, Sun et al. (11) showed that anti-CD40L mAb treatment had no effect in preventing GAD development without concomitant CTLA4-Ig treatment in a mouse aortic transplant model.
Using CD40L deficient mice, we therefore investigated the mechanisms by which blockade of CD40-CD40L interaction induce long-term allograft survival in a murine heart transplantation model, and evaluated long-term allografts for the development of GAD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male wild-type (WT) C57BL/6 (B6; H-2b) or BALB/c (B/c; H-2d) mice and male B6 CD40L-deficient (CD40L-/-) mice aged 8–12 wk and weighing 20–25 g were used as recipients or donors. CD40L-/- mice at least 10th generation backcrossed on the B6 background, were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 and B/c WT mice were obtained from Taconic Farms (Germantown, NY) or The Jackson Laboratory. C3H/HeJ (C3H, H-2k) mice were obtained from The Jackson Laboratory. The mice were maintained in the Harvard Medical School animal facilities on acidified water. Sentinel animals in the same room were surveyed serologically and were consistently negative for viral pathogens. All experiments conformed to approved animal care protocols.
Vascularized heterotopic cardiac transplantation
Allografts were vascularly anastomosed in an intraabdominal location using the technique described by Corry et al. (12), as modified by Nagano et al. (13). Graft ischemic time was typically 20–25 min and total operative time was 45–50 min with a success rate (beating hearts) of more than 90%. All grafts were evaluated daily by measuring the force of palpable heart beat, adopting a scoring system with a scale of 0–4 as described by Corry et al. (12). Rejection of heart grafts was not associated with death of recipients.
Skin transplantation
Full-thickness tail skin grafts (1 cm2)
were transplanted onto the thorax of recipient mice, stitched with 4-0
Ethibond (Johnson & Johnson. Somerville, NJ) at the four corners, tied
over with 4-0 Ethibond, and secured with a Silkypore (Tokyo Eizai Lab,
Tokyo, Japan) for 7 days. Rejection was defined as the complete loss of
viable graft tissue.
Graft harvest
Harvested heart allografts were transversely sectioned into three parts; the basal section was fixed with 10% phosphate buffered formalin and embedded in paraffin for morphological examination, the mid-portion was frozen in OCT compound (Tissue Tek; Miles, Elkhart, IN) and stored at -80°C for immunohistochemical staining, and the apical portion was used for intracellular cytokine staining of lymphocytes using flow cytometry or RNase protection assay (RPA) (4).
Histological evaluation
For assessment of parenchymal rejection (PR) and GAD, grafts were analyzed in a blinded fashion using sections stained by hematoxylin and eosin, and elastic tissue stains, as described previously (13). The severity of PR was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no rejection; 1, focal mononuclear cell infiltrates without necrosis; 2, focal mononuclear cell infiltrates with necrosis; 3, multifocal infiltrates with necrosis; 4, widespread infiltrates with hemorrhage and/or vasculitis) (13, 15). The overall GAD score was averaged from the scores of all epicardial and intramyocardial arteries and arterioles in each graft (0, vascular stenosis <10%; 1, 10–25% stenosis; 2, 25–50% stenosis; 3, 50–75% stenosis; 4, > 75% stenosis), as described previously (13). Typically, 10 or more vessels were graded in each graft; scores were expressed as the mean ± SD.
Extraction of lymphocytes from spleen and cardiac allograft
Spleens 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 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. Lymphocytes were washed twice more in PBS, and resuspended in RPMI with 10% FCS supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 57 µM 2-ME (C/10 medium) at a concentration of 1 x 107 cells/ml.
Sections from harvested cardiac allografts were minced with a sterile blade and incubated in 10 ml buffered saline with 2% BSA and 2 mg/ml collagenase at 37°C for 2 h. The cells were strained through a 70 µM nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Lymphocytes were isolated from these cells by Ficoll (Organon Teknika, Durham, NC) density gradient centrifugation for 20 min at 800 rpm. After washing twice in RPMI 1640, lymphocytes were resuspended in C/10.
Antibodies
All of the Abs used for T cell proliferation assay, cytokine
ELISA, and flow cytometry analysis were purchased from PharMingen (San
Diego, CA). For T cell proliferation assay, anti-CD3 mAbs (Hamster
IgG, clone 145-2C11) were used. For intracellular cytokine staining,
extracted cells were stained with either a biotinylated rat IgG1
(R3-34) as isotype-matched control or biotinylated anti-IFN-
(XMG1.2) or biotinylated anti-TNF- (MP6-XT). For CD28, CTLA-4,
or CD80 staining, extracted cells were stained with either a
PE-conjugated hamster Ig (anti-TNP; 2,4,6-trinitrophenol) as
isotype matched control or anti-CD28-PE (37.51), anti-CTLA-4-PE
(9H10), or anti-CD80-PE (16-10A1). For CD40 and CD86 staining,
extracted cells were stained with either a PE-conjugated rat IgG2a
(R35-95) as isotype-matched control or anti-CD40-PE (3/23) or
anti-CD86-PE (GL1). For CD4, CD8, or CD11b staining,
anti-CD4-PerCP (RM4–5), anti-CD8-PE (53-6.7) or
biotinylated-anti-CD8 (53-6.7) and streptavidin-APC as second Ab,
and anti-CD11b-FITC (M1/70) were used. For ELISA, purified or
biotinylated anti-IFN- (rat IgG1, clone XMG1.2), or
anti-TNF- (rat IgG1, clone MP6-XT3) were used.
For Ig ELISA or measurement of serum alloantibodies using flow cytometry, purified anti-IgG (H+L) or IgM, alkaline phosphatase-conjugated anti-IgG (H+L) or IgM mAbs, or FITC- conjugated anti-IgG (H+L) or IgM mAbs were used (Southern Biotechnology Associates, Birmingham, AL). For CD40 stimulatory mAb treatment experiment, CD40L-/- transplant recipients were treated with anti-CD40 mAbs (rat IgG2a, clone 3/23), which was a generous gift of Drs. Alex Macadam and Arlene Sharpe (Brigham and Women’s Hospital, Boston, MA).
T cell proliferation assays
One-way MLR and immobilized anti-CD3 Ab-induced T cell proliferation assays were performed using whole naive splenocyte populations of WT and CD40L-/- B6 or B/c primed splenocytes harvested from WT or CD40L-/- B6 recipients 4 wk following transplant. Fifty microliters (5.0 x 105 cells) each of responder B6 splenocytes and irradiated (30 Gy) stimulator B/c or C3H/HeJ (C3H; H-2k) splenocytes were added in quadruplicate to 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA). Alternatively, 5.0 x 105 responder B6 splenocytes were cultured in 96-well microtiter plates precoated with anti-CD3 mAb (1 µg/ml). Cells were cultured at 37°C under 5%CO2 atmosphere for 1–5 days; proliferation at each time point was assessed by pulsing the wells for 6 h with 1 µCi of tritiated thymidine (New England Nuclear, Boston, MA). Proliferation was measured as incorporated radioactivity (cpm) using a Betaplate scintillation counter (LKA Pharmacia). Results were expressed as the mean ± SEM.
Cytokine ELISA
Sandwich ELISA was performed using paired Abs (anti-IFN-
and biotinylated anti-IFN- mAbs (PharMingen), and
streptavidin-HRP). Briefly, 50 µl of purified anti-cytokine Abs
(2 µg/ml) were coated overnight on 96-well microtiter plates (Becton
Dickinson, Mountain View, CA) at 4°C. After blocking the plates with
2% BSA in PBS at room temperature for 1 h, 50 µl of the sample
supernatant (diluted 1:2) from MLR culture was added followed by
incubation for 6 h at room temperature. The plates were washed
with PBS containing 0.01% Triton-X and then reacted with 50 µl of
biotinylated anti-cytokine Abs (1 µg/ml) for 45 min. After
washing six times with PBS containing 0.01% Triton X, plates were
incubated with peroxidase-labeled streptavidin for 30 min, followed by
addition of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
(Sigma, St. Louis, MO) for colorimetric reaction. Absorbance data (at
414 nm) were collected using microplate reader Emax (Molecular Devices
Corporation, Sunnyvale, CA). Standard curves for each cytokine were
generated using recombinant cytokines (rIFNg; Biosource International,
Camarillo, CA).
Ig ELISA
Sandwich ELISA was performed using paired Abs (purified anti-IgG(H+L) or IgM, and alkaline phosphatase-conjugated anti-IgG(H+L) or IgM mAbs. Briefly, 100 µl of purified anti-Ig Abs (5 µg/ml) were coated overnight on 96-well microtiter plates (Becton Dickinson) at 4°C. After blocking the plates with 2% BSA in PBS at room temperature for 1 h, 100 µl of the sample serum (serially diluted 1:3) were added followed by incubation for 2 h at room temperature. The plates were washed with PBS containing 0.01% Triton X and then incubated with 100 µl of alkaline phosphatase-conjugated anti-Ig Abs (1 µg/ml) for 90 min. After washing six times with PBS containing 0.01% Triton X, plates were incubated with developing reagent, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma), for 30 min. Absorbance data (at 405 nm) were collected using microplate reader Emax (Molecular Devices).
Cell surface immunofluorescent staining
Cell surface staining and flow cytometry were performed using the methods described by Stinn et al. (14). Briefly, extracted cells were incubated with FITC, PE, or PerCP conjugated surface marker Abs, anti-CD4, CD8 (2 µg/ml), or CD11b mAbs (1 µg/ml) or PE conjugated costimulatory molecules Abs, anti-CD80, CD86, CD28, or CTLA4 Abs (2 µg/ml) for 30 min at room temperature, followed by washing in PBS, and fixation in 1% paraformaldehyde in PBS.
Intracellular cytokine staining
Intracellular cytokine staining and flow cytometry were performed using the methods described by Stinn et al. (14). Briefly, extracted cells were stimulated with 25 µM ionomycin (Sigma), and 10 ng/ml PMA (Sigma) for 4 h at 37°C under a 5% CO2 humidified atmosphere. Brefeldin A (10 µg/ml, Sigma) was added for the duration of the culture to block cytokine secretion and thereby improve detection. After stimulation, cells were centrifuged at 1200 rpm for 5 min, and washed in ice-cold PBS before fixing at room temperature with 4% paraformaldehyde in PBS for 10 min. For intracellular staining, cells were permeabilized with saponin/PBS buffer (0.5% saponin (Sigma), 1% BSA, 0.1% NaN3 in PBS). After incubation with 0.25 µg Fc block (PharMingen) for 5 min, cells were labeled with 10 µg/ml of a primary biotinylated anti-cytokine Ab or biotinylated isotype-matched control Ab (PharMingen) for 30 min at room temperature. After washing twice with saponin/PBS buffer, the cells were incubated with APC-conjugated streptavidin (2.0 µg/ml) for 30 min at room temperature. The cells were washed twice with saponin/PBS buffer, washed with PBS alone to seal the membranes, and stained with FITC-, PE-, or PerCP-conjugated surface marker Abs (2.5 µg/ml) for 30 min at room temperature, followed by washing in PBS.
Flow cytometric analysis
Flow cytometry was performed on a four color FACScan flow cytometer (Becton Dickinson) using CellQuest software. Dead cells and polymorphonuclear cells were excluded on the basis of light scatter characteristics. Scatter regions for infiltrating mononuclear cells were established before each collection using stimulated splenocytes. Collection of cytokine staining data from allografts was restricted to this gated region. In total, 10,000 events within the mononuclear cells scatter region were routinely analyzed.
Total RNA extraction and RPA
Total RNA was isolated from cardiac allografts with TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. The concentration of RNA was determined by ultraviolet spectroscopy at 260 nm. The RPA was performed according to the manufacturer’s recommendations using 20 µg of total RNA from each grafted heart and mck-1 or mck-3b Riboquant multiprobe RPA kits (PharMingen). After electrophoresis on 6% polyacrylamide/urea gel, the patterns of radioactivity were captured by phosphorimager plates (Molecular Dynamics). The data were recorded as intensity of OD (IOD) using a Molecular Dynamics PhosphorImager. The cytokine mRNA IOD was normalized to the GAPDH gene mRNA IOD using ImageQuant software.
Measurement of serum alloantibodies
Naive splenocytes (1 x 106) of B/c WT animals, prepared as described above, were incubated for 30 min at room temperature with 30 µl of 1:10 diluted sera obtained from naive B6 mice (control), or WT or CD40L-/- B6 recipients of heart transplants, 4 wk after grafting. Cells were washed twice, incubated with FITC-conjugated anti-mouse IgG (H+L) or IgM (Southern Biotechnology Associates), and PE-conjugated anti-CD8 and PerCP-conjugated anti-CD4 Abs (PharMingen). Flow cytometry was performed on a three color FACScan flow cytometer (Becton Dickinson) using CellQuest software.
Statistical analysis and transplant survival analysis
Comparative analysis of cardiac graft survival was accomplished via the Kaplan-Meier cumulative survival method and survival differences between two groups were determined using the log-rank (Mantel-Cox) test. Comparisons between two groups for cytokine and Ig ELISA, MLR, T cell proliferation, and normalization of RPA data, parenchymal rejection and graft arterial disease scores, graft-infiltrating cell number, and phenotype distribution rate were accomplished by one-way ANOVA. Comparison over the time course of parenchymal rejection and graft arterial disease scores and normalization of RPA data were analyzed by ANOVA for repeated measures using StatView 4.5 for Macintosh (Abacus Concepts, Berkeley, CA). Data are expressed as mean ± SE (SEM). p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascularized heterotopic abdominal cardiac transplantations were
performed using total allogeneic mismatched combinations of BALB/c
(H-2d) (B/c) and WT or
CD40L-/- C57BL/6J (H-2b)
(B6) mice. WT B6 donor hearts were rejected by 13.1 ± 4.9 days
when transplanted into WT B/c recipients (n = 11);
CD40L-/- B6 donor hearts were rejected by
13.0 ± 4.6 days in WT B/c recipients (n = 6; no
significant difference) (Fig. 1
A). B/c hearts in B6 hosts
ceased functioning by 8.4 ± 1.4 days (n = 11). In
contrast, all B/c donor hearts in
CD40L-/- B6 recipients maintained good (4+
palpation) graft function for more than 12 wk (n = 8)
(p < 0.0001) (Fig. 1B).
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By postoperative day 7, the histologic grade of PR in WT B/c
allograft hearts was significantly greater for B6 WT than for B6
CD40L-/- recipients (Fig. 2A, and compare with Fig. 2, D and E). PR scores were 3.13 ± 0.52 and
2.17 ± 0.41 (p = 0.0028) in WT
(n = 8) and CD40L-/-
(n = 6) recipient allografts, respectively. The extent
of PR did not significantly change in CD40L-/-
recipient allografts with time (Fig. 2B, D–G)
although there was a trend toward diminishing infiltrates. Because
grafts in WT hosts failed within 2 wk of transplant, they did not
develop the intimal hyperplastic lesions characteristic of GAD.
However, the long-term grafts in CD40L-/- hosts
showed progressively increasing severity and extent of GAD (Fig. 2, C and H).
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We assessed the ability of CD40L-/-
lymphocytes to respond to direct TCR ligation and allogeneic
stimulation in primary MLR. CD40L-/-
lymphocytes and WT cells stimulated by immobilized anti-CD3 mAbs
displayed the same [3H]thymidine incorporation
(Fig. 3A), indicating that
CD40L-/- T cells have normal proliferative
capacity. Although WT lymphocytes had greater proliferation than
CD40L-/- lymphocytes in a primary MLR,
CD40L-/- lymphocytes still exhibited a strong
allogeneic response with similar kinetics (Fig. 3B). We also
examined the levels of IFN- and IL-4 expression in supernatants from
anti-CD3 stimulated cultures or primary MLRs, at 1–5 days after
plating. Levels of IFN- were comparable between
CD40L-/- and WT responder cells in either
anti-CD3 or MLR cultures (Figs. 3, D and E).
No IL-4 was detected in any of these cultures (data not shown).
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In MLR using in vivo primed splenocytes harvested 4 wk
posttransplant from WT or CD40L-/- recipients,
WT lymphocytes had significantly (p < 0.0001)
higher [3H]thymidine uptake than
CD40L-/- lymphocytes (Fig. 3
B).
IFN- secretion by WT responder cells was also significantly
increased on day 1 through day 5 compared with
CD40L-/- B6 responder cells (Fig. 3E). Again, no IL-4 was detected (data not shown).
To assess whether the mechanism of long-term allograft survival in
CD40L-/- recipient allografts was due to the
development of donor Ag specific tolerance, we compared the primary MLR
and the B/c heart transplant-primed MLR to third party C3H stimulators.
As shown in Fig. 3C, [3H]thymidine
incorporation was comparable between the primary MLR to C3H and the MLR
to C3H using B/c-primed responder splenocytes. These data demonstrate
donor-specific hyporeactivity.
Second set skin transplantation demonstrates allospecific tolerance of CD40L-/- heart allograft recipients
To confirm the induction of donor-specific tolerance, we performed
skin transplantation after heart transplantation. It has been
demonstrated that CD40L blockade alone does not prevent allograft skin
rejection (10). Similarly, skin allografts are rejected in
naïve CD40L-/- hosts (Fig. 4A). However, B6
CD40L-/- hosts with long-term surviving B/c
cardiac allografts retained B/c skin grafts for more than 4 wk (Fig. 4, B and C). The third party (C3H) skin grafts were
rejected within 2 wk of transplant (Fig. 4D), demonstrating
the induction of allospecific tolerance.
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The long-term survival of allografts in
CD40L-/- recipients could be due to lack of
signaling via CD40 rather than via CD40L. Thus, to test whether
ligation of allograft CD40 may induce allograft rejection even in
CD40L-/- recipients, we performed the following
study. B/c to CD40L-/- B6 cardiac transplant
recipients were divided into two groups; group 1 (n =
3) received intraperitoneal injection of 100 µg/day of activating
anti-CD40 Ab (3/23) for 7 days beginning at day 0 (operative day),
following a protocol previously shown to be stimulatory
(16). Group 2 (n = 5) received i.p.
injection of 100 µg/day of isotype matched rat IgG2a for 7 days
beginning at day 0 (operative day). Efficacy of anti-CD40 Abs
treatment was confirmed by an increased serum IgG on postoperative day
10 (Fig. 5). Hearts functioned well in
both groups with comparable beat strength at 4 wk. All donor hearts
were harvested on postoperative week 4, and intracellular cytokine
staining and histological studies were performed. There was no
significant difference in the levels of PR and GAD score between the
two groups (Table I). The apparent lower
GAD score from this set of animals at 4 wk, relative to Fig. 2C is not statistically significant. The results suggest
that ligation of CD40 expressed on cells of the donor heart does not
play an important role in allograft rejection.
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To test whether long-term allograft survival in
CD40L-/- recipients corresponded to differences
in inflammatory infiltrates, we stained cells extracted from grafts
with anti-CD4, CD8, CD11b, NK1.1, and B220 mAbs (14),
and analyzed them by flow cytometry. Corresponding to the decreased PR
grade, the total number of the infiltrating mononuclear cells was less
in allografts harvested 6 days after transplant from
CD40L-/- recipients vs WT recipients (0.8
x 106 and 1.5 x 106
cells/graft, respectively; p < 0.005). Nevertheless,
the relative distributions of CD4+,
CD8+ and CD11b+ cells were
comparable (Table II).
NK1.1+ cells and B220+ B
cells represented <1% in both CD40L-/- and WT
recipient allografts (data not shown).
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To test whether CD40L deficiency affects cytokine profiles in the
allograft hearts, cytokine mRNA expression at postoperative day 6 was
analyzed by RPA. Total RNA (20 µg) from each grafted heart was
hybridized with 32P-labeled cytokine mRNA probes
(Fig. 6, A and C).
RPA data were normalized to the GAPDH gene mRNA to permit quantitation
of the various mRNAs (Fig. 6, B and D). There was
no detectable IL-4 or IL-5 mRNA in either
CD40L-/- or WT recipient allografts (Fig. 6A). There was significantly lowered expression of IL-2 and
IL-10 mRNA in CD40L-/- vs WT recipient
allografts, whereas there was no significant difference in IL-15 mRNA
expression. IL-6 mRNA expression was also reduced significantly in
CD40L-/- recipient allografts
(p < 0.0001). The largest difference between
CD40L-/- and WT recipient allografts was in the
expression of IFN- (p < 0.0001). IFN-
mRNA was strongly expressed in WT recipient allografts, whereas being
significantly reduced in CD40L-/- recipient
allografts. TNF-ß expression was significantly reduced in
CD40L-/- relative to WT recipient allografts
(Fig. 6, C and D) (p <
0.05). Although there was no significant difference in TGF-ß2 mRNA
expression between CD40L-/- and WT recipient
allografts, TGF-ß1 mRNA expression was significantly reduced in
CD40L-/- hosts (p <
0.0001). Likewise, TNF- mRNA expression was significantly reduced in
CD40L-/- and WT recipient allografts
(p < 0.0001). Notably, although some cytokines
were reduced in CD40L-/- recipients relative to
WT hosts commensurate with decreased cell infiltrate, the overall
cytokine profiles were not qualitatively different between the two
types of recipients.
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; n = 4 for each
time point). All cytokines showed a progressive and significant
decrease with time (repeated ANOVA); no IL-4 or IL-5 mRNA was seen at
any time point.
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To determine the cell source of IFN- and TNF-,
graft-infiltrating cells harvested on postoperative day 6 were analyzed
by intracellular cytokine staining. Three color staining, using
anti-CD4, anti-CD8, and anti-CD11b, was used to gate on the
three populations previously defined. The fourth color was then used to
analyze the cytokines (IFN- or TNF-). IFN- and TNF-
expression by both CD4+ and
CD8+ T cells was significantly reduced in
CD40L-/- recipient allografts compared with WT
recipient allografts (Fig. 8). TNF-
expression by graft-infiltrating CD11b+ cells was
also diminished in CD40L-/- recipients compared
with WT recipients. No IL-4 expression was detected in either
CD40L-/- or WT recipient allografts (data not
shown).
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Cardiac allografts contained numerous infiltrating mononuclear cells, including APCs such as monocytes, macrophages, and B cells, all of which express costimulating molecules such as CD80 (B7-1), CD86 (B7-2), and CD40. We hypothesized that a lack of CD40-CD40L interaction might prevent generation of allograft immunity by failing to increase B7 expression and/or by modulating the expression of CTLA-4 or CD28.
In vitro culture of B/c splenocytes with
CD40L-/- or WT B6 stimulators resulted in
comparable expression of CD80, CD86, CD28, and CTLA-4 (data not shown).
Flow cytometry for the same costimulating molecules was also performed
on infiltrating cells from B/c to WT or B/c to
CD40L-/- recipient allografts harvested on
postoperative day 6. CD40, CD80, and CD86 levels on
CD11b+ mononuclear cells were all lower in
CD40L-/- recipient allografts than in WT
recipient allografts (Fig. 9A). Interestingly, CD40,
CD80, and CD86 levels on CD4+ cells and
CD8+ cells were all slightly higher or unchanged
in CD40L-/- recipient allografts relative to WT
recipient allografts (Fig. 9A). The surface expression of
CTLA-4 on both CD4+ and
CD8+ T cells was comparable in both WT and
CD40L-/- recipient allografts (Fig. 9B). CD28 expression on graft-infiltrating
CD8+ cells was higher for WT recipients than for
CD40L-/- recipient allografts, although CD28
expression from CD4+ cells was comparable (Fig. 9B).
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Due to the absence of CD40L, CD40L-/- mice
do not class-switch their Ab isotypes and have elevated IgM levels. To
test whether the isotype of recipient alloantibody correlated with
long-term graft survival in CD40L-/- hosts, we
incubated B/c splenocytes with serum harvested from B6 WT or B6
CD40L-/- recipients of B/c hearts. Bound
alloantibodies were then detected by FITC-conjugated anti-mouse IgG
or IgM and flow cytometry. As shown in Fig. 10, by 4-wk posttransplant, IgG Abs
reactive with donor CD4+ T cells (and
CD8+ T cells, not shown) were present in WT
allograft recipients but were weakly expressed, if at all, in
CD40L-/- hosts. IgM alloantibodies were
comparably generated in both WT and CD40L-/-
recipients. Staining for Ig in CD40L-/-
hosts 4-wk posttransplantation was negligible (not shown), indicating
that the circulating Abs detected by flow cytometry are not being
affected by any intragraft accumulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Larsen et al. (10) showed that anti-CD40L mAb
could prevent murine cardiac allograft rejection, and combinations of
anti-CD40L mAb and CTLA4- Ig initiated at the time of
transplantation were synergistic, leading to long-term survival.
Although anti-CD40L mAb alone did not affect the expression of
T cell cytokines or of B7 molecules, the combination of CTLA4 Ig plus
anti-CD40L inhibited the expression of IL-2, IL-4, IL-10, and
IFN-, as determined by RT-PCR. Kirk et al. (18) also
demonstrated that anti-CD40L mAb treatment allowed long-term renal
allograft survival in monkeys and long-term survivors lost their mixed
lymphocyte reactivity in a donor-specific manner. In those experiments,
the relative role of CD40 or CD40L on donor or recipient cells could
not be ascertained. Here, we unambiguously demonstrate that absence of
CD40L on host cells is responsible for the graft survival. That T
cells from CD40L-/- and WT mice are comparably
stimulated by anti-CD3 treatment indicates that CD40L depletion per
se does not cause global T cell unresponsiveness.
Induction of donor-specific tolerance has become a major goal for innovation in organ transplantation. This study examined potential pathways by which host CD40L depletion could result in allograft survival and promote allospecific tolerance. In WT hosts 4 wk after transplantation, the MLR to donor B/c allogeneic cells increased significantly relative to the primary MLR before transplantation. In contrast, the MLR of CD40L-/- recipients to donor allogeneic B/c cells decreased relative to the primary MLR before transplant. The decreased responses were donor-specific because response to third-party cells (C3H) remained unchanged before and after transplantation. Similarly, second set B/c skin grafts in CD40L-/- recipients of B/c heart allografts were retained, whereas third-party (C3H) skin grafts were promptly rejected. Thus, cardiac transplantation into CD40L-/- hosts results in allospecific tolerance, comparable to that seen using anti-CD40L mAbs (10, 18).
There are two possible mechanisms by which absence of CD40L may induce tolerance and promote long-term cardiac allograft survival. First, CD40L may interact with CD40 on donor heart endothelial cells and myocytes to induce events that cause endothelial cell and myocardial dysfunction, and eventual graft failure. A second possibility is that CD40L may be a critical receptor for T cell costimulation via CD40 on APCs. In this case, depletion of T cell CD40L may result in reduced T cell activation or T cell cytokine production, and even T cell anergy.
To examine the first hypothesis, we treated
CD40L-/- allograft recipients with 3/23
anti-CD40 stimulatory Abs. Although cytokine expression (IFN-
and TNF-) by T cells was relatively increased using 3/23 (data not
shown) and 3/23 induced recipient Ig class switching, the allografts
were not rejected. These results indicate that CD40 ligation directly
on either host or donor cells does not play a role in allograft
failure.
Instead, the present study supports the second hypothesis that
long-term graft survival in CD40L-/- hosts is
associated with diminished T cell activation and subsequent effector
functioning. Besides overall reduced inflammatory infiltrates, IFN-
and TNF- mRNA expression were significantly reduced in
CD40L-/- recipient allografts. In addition,
TNF-ß, IL-2, IL-6, IL-10, and TGF-ß1 mRNA were also reduced in
CD40L-/- hosts. Flow cytometry shows that the
reduction in these cytokines was due to diminished production by both
CD4+ and CD8+
graft-infiltrating T cells.
Recently, Hancock et al. (19) showed that prolongation of
graft survival by anti-CD40L mAb was accompanied by inhibition of
Th1 cytokine (IL-2 and IFN-) and up-regulation of Th2 cytokines
(IL-4 and IL-10). They suggested that Th1 to Th2 immune deviation was a
principal mechanism by which blockade of CD40-CD40L interactions
induced long-term allograft survival. In experimental and clinical
transplantation, rejection has often appeared to correlate with the
detection of Th1 rather than Th2 cytokines (19). Such
observations are the basis of the Th1/Th2 paradigm which predicts that
if rejection correlates with Th1 dominant cytokines, Th2 induction may
result in graft tolerance (20). This contrasts with our
data, showing that blockade of CD40L reduced Th1 cytokines (IL-2 and
IFN-), as well as IL-10, and no IL-4 and IL-5 (Th2 cytokines) were
detected in either WT or CD40L-/- recipient
allografts. Thus, although the Th1/Th2 paradigm is attractive, the
available data do not support such a clear distinction. Indeed, the
acute failure of allogeneic hearts in
IFN--/- mice (13) or
IL-4-/- mice (21) appear to rule
out a strict requirement for either of the cytokines in graft
rejection. In fact, IFN- and IL-4 may both play important roles in
long-term allograft survival (22, 23).
TNF or IL-6 have been implicated as major effectors of graft failure in
rejection (24, 25, 26, 27). Elevated TNF may cause neutrophil and
endothelial activation and induce the expression of adhesion molecules
on endothelial cells resulting in accumulation of leukocytes in the
transplanted organ (25). Coito et al. (28)
demonstrated that anti-TNF Abs prevented allograft rejection in
murine heart transplantation. Russell et al. (29) also
demonstrated that gene transcript levels for IL-6 in rat cardiac
allografts increased significantly as compared with syngeneic grafts.
In our study, elevation of TNF and IL-6 mRNA during transplantation
declined significantly in CD40L-/- recipient
allografts. Our flow cytometry results also demonstrated greatly
reduced expression of TNF- by CD4+ and
CD8+ T cells in CD40L-/-
recipient allografts. Thus, reduced TNF and IL-6 production may be
important for acute allograft survival.
Another potential mechanism by which blockade of CD40-CD40L could prevent transplant rejection is altering B7-CD28 costimulation pathways (10). Several reports suggest that a primary role of activated Th cells in the generation of CD4-dependent cell-mediated immunity was to provide CD40 ligation on APC presumably to increase B7 expression (30, 31, 32). Interactions between B7 family (CD80 and CD86) and CD28 increase TCR signals and synergize with various cytokines (IL-4, IL-6, and IL-10). Anti-CD86 mAbs can block MLR and stimulation of T cells by APCs (33) and allografts in recipient mice deficient in both B7-1 and B7-2 functioned for at least 12 wk (34).
Confirming prior work (35), we found no difference in costimulator expression in MLR with WT or CD40L-/- stimulators (data not shown). However, intragraft CD11b+ cells have lower CD80 and CD86 expression in CD40L-/- recipient allografts, relative to WT recipient allografts. Interestingly, CD80 and CD86 expression by CD4+ and CD8+ cells were either unchanged or increased in the CD40L-/- recipient allografts, compared with WT allografts. These results agree with the previously reported finding that total CD80 and CD86 transcripts in cardiac allografts were not reduced by blockade of the CD40 pathway (36). We suggest, therefore, that the reduced expression of CD80 and CD86 by CD11b+ cells in CD40L-/- recipient allografts is most relevant. Interestingly, even in the setting of reduced CD80 and CD86 expression on CD11b+ APC, cell surface expression of CTLA-4 by CD4+ and CD8+ cells was comparable in WT and CD40L-/- recipient allografts. Moreover, CD28 expression on CD4+ cells was similar in WT and CD40L-/- recipient allografts, although CD8+ cells from CD40L-/- hosts showed lower CD28 levels. Taken together, reduced B7 expression on macrophage APC may be an important pathway by which blockade of CD40-CD40L signaling induces tolerance. Consistent with this hypothesis, Tan et al. (3) demonstrated that the B7 antagonist, CTLA4-Ig blocked the human mixed lymphocyte reaction and induced Ag-specific unresponsiveness.
The present experiments also evaluated the activation status of the graft-infiltrating cells by flow cytometry at 1 wk after transplant (data not shown). Comparable levels of CD44 and CD69 (very early activation Ag) were seen in both WT and CD40L-/- recipient allograft and no difference was seen in the levels of CD62L on CD4+ or CD11b+ cells (data not shown). Thus, at least by surface marker criteria, absence of CD40L on host cells did alter initial T cell activation status.
The interaction between CD40 on B cells and CD40L on T cells plays an important role in B cell activation, including proliferation, the generation of a thymus-dependent Ab response and Ig isotype switching (37, 38, 39). Buhlmann et al. (40) showed that in vivo administration of a mAb specific for CD40L (MR1) diminished the response to alloantigen on B cells in vitro. Immunization with resting B cells from CD40 knockout mice also induced tolerance to alloantigen (41). B cell responses are potentially relevant in GAD pathogenesis because Hancock et al. (9) reported that IgG alloantibodies can induce accelerated arteriosclerosis after transplant.
Our work found no IgG alloantibodies in CD40L-/- recipients of B/c heart allografts, suggesting that lack of Ig class-switching may be relevant to the long-term survival in CD40L-/- recipients. Nevertheless, IgG Abs are not the only explanation, because induction of IgG by 3/23 anti-CD40 mAbs did not induce allograft failure. Moreover, despite induction of tolerance and long-term allograft survival, and in the absence of allospecific IgG, the hearts in CD40L-/- recipients still developed GAD.
Our findings support the view that CD40L on host cells plays an important role in the regulation of allogeneic responses and induction of donor-specific tolerance to cardiac allografts in the mice. This outcome is potentially mediated by regulation of accessory molecule expression on APC, and is also associated with reduced cytokine expression and lack of IgG allo-specific Abs. More importantly, however, the early transient parenchymal rejection which may be required to induce allograft tolerance (42, 43), likely triggers a cascade of Ag nonspecific effectors which will culminate in chronic vascular lesions and eventually lead to ischemic allograft failure. Although combined treatment with CTLA4-Ig and anti-CD40L mAb did ameliorate GAD in an aortic allograft model (11), this may not be attributable to a unique beneficial effect of anti-CD40L treatment but rather due to complete immunologic blockade, as we have seen with chronic anti-CD4 and anti-CD8 treatment (17). Similarly, concomitant anti-CD40L mAb and donor cell administration led to long-term survival without GAD in a mouse heart transplant model; however, the relative contribution of donor cells vs CD40L blockade cannot be assessed (9). Thus, development of tolerance alone may be insufficient to insure successful indefinite graft functioning; it is also critical to address the underlying mechanisms of graft arteriosclerosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard Mitchell, Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Ave, LMRC 515, Boston, MA 02115.
3 Abbreviations used in this paper: CD40L, CD40 ligand; WT, wild type; GAD, graft arterial disease; RPA, RNase protection assay; PR, parenchymal rejection.
Received for publication November 22, 1999. Accepted for publication June 29, 2000.
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