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Division of Liver and Pancreas Transplantation, Department of Surgery, Dumont-University of California Transplant Center, University of California School of Medicine, Los Angeles, CA 90095
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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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We have long been interested in developing new strategies to manage sensitized transplant recipients, and over a decade ago established a rat model in which skin graft-induced sensitization triggers accelerated (24–36 h) rejection of vascularized cardiac allografts (3). As distinct from hyperacute rejection, which develops within minutes or hours and associates with high levels of pre-formed Abs to the donor, resulting in a classical picture of interstitial hemorrhages/vascular thrombosis, studies of accelerated rejection have suggested that both T and B cell responses may be important (1, 3, 4). We have recently developed a model of accelerated rejection of cardiac allografts in sensitized mice to further dissect the cascade of host cellular and humoral immune responses and to exploit a broader range of mAbs and the multitude of genetically deficient strains available (5). Although multiple effector pathways are involved, our studies have clearly shown that T cell-dependent mechanisms alone can mediate accelerated (<48 h) rejection of cardiac allografts.
T cell activation is dependent on interlocked signaling events that occur through engagement of cell surface receptors. In addition to the recognition of the MHC peptide complex by the TCR, efficient T cell activation and subsequent induction of effector functions require a second costimulation signal (6, 7). It is now generally accepted that CD40-CD154 interactions may provide such a signal during T cell activation, T cell-B cell interactions, and endothelial activation (8). CD154 (CD40 ligand), a member of the TNF family, is expressed predominantly on mature activated CD4+ and some CD8+ T cells, and interacts with CD40 expressed by APCs and B cells (9, 10, 11). Although the importance of CD154-mediated CD4+ T cell interactions with B cells is well recognized, it has been less clear what role CD154 may play in CD8+ T cell activation (12). The accruing data suggest that CD154-CD40-mediated activation of dendritic cells (DCs)2 by Ag-specific CD4+ T cells is essential for subsequent priming of CD8+ T cells (13, 14, 15). Thus, with T cell help needed to generate a productive CD8 T cell response, triggering of DCs by CD4 T cells was required before DC encounter with Ag-specific CD8+ T cells. Although activated CD8+ T cells can express CD154 (16, 17), little is known of putative functional roles and feedback interactions between CD8+ T cell CD154 and CD40 expressed by APCs. Recent evidence points toward a critical role of CD154 in the optimal generation of the mucosal CD8+ T cell response (18).
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 (19, 20, 21). This highlights the role of the CD40-CD154 costimulation pathway in the immune cascade leading to acute allograft rejection. However, relatively little is known of the role of CD154 costimulatory signals in the mechanism of host sensitization and whether their blockade may affect graft rejection in sensitized hosts. By employing CD154-/- mice as recipients of sequential skin and cardiac allografts and by targeting CD154-CD40 interactions in wild-type (WT) skin-sensitized hosts, we have recently documented an essential role of the CD154 pathway in host sensitization to alloantigen and identified CD8+ T cells as principal targets of the CD154 blockade in that model (22). Unlike preventive anti-CD154 mAb treatment in the sensitization phase (i.e., between skin and cardiac engraftment), peri-transplant CD154 blockade during the effector phase (i.e., at the time of cardiac engraftment) failed to protect heart transplants from accelerated rejection. The present study was aimed at dissecting putative mechanisms of CD154 costimulation blockade-resistant allograft rejection and improving the efficacy of this novel therapeutic strategy in sensitized recipients. We analyzed CD8+ T cells, principal targets of the CD154 blockade, in their costimulation requirements for activation in either naive or Ag-primed states. To the best of our knowledge, this study is the first to provide evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed memory CD8+ T cells, and 2) successful treatment of sensitized recipients by adjunctive CD154 costimulation blockade protocols to achieve stable and long term graft acceptance in a clinically relevant transplantation model.
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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), CBA/Ca (CBA; H-2k) male mice and CD154-deficient (CD154-/-; B6) male mice (intercrossed at least 10 generations), aged 8–12 wk (20–25 g), were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in the University of California, Los Angeles animal facilities under specific pathogen-free conditions.
Transplantation
Orthotopic full-thickness skin grafts (
0.5 cm in diameter)
from B/c donors were sutured bilaterally onto the flanks of prospective
WT or CD154-/- B6
recipients. These recipients were then challenged 10 or 40 days later
with intraabdominal heterotopic B/c heart transplants. 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 selective pathological examination.
Treatment
Anti-mouse CD154 mAb (MR1; Bioexpress, West Lebanon, ME) was administered at cardiac engraftment (0.5 mg/mouse i.v.). Control recipients were treated with relevant doses of hamster Ig. Rat anti-mouse depleting CD8 (2.43; courtesy of Dr. H. Auchincloss, Massachusetts General Hospital, Harvard Medical School, Boston, MA) or CD4 (GK1.5; BD PharMingen, San Diego, CA) was administered at either 0.25 (CD8) or 0.5 (CD4) mg/mouse/day, respectively, i.v. for 3 consecutive days (days -2, -1, and 0 of cardiac engraftment). Control animals were given relevant doses of rat Ig. In CTL induction experiments in vivo, MR1 (0.5 mg/mouse) or murine CTLA4Ig (0.25 mg/mouse) was used i.v. at the time of skin grafting.
In vitro MLR
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 then resuspended in 5 ml
Tris-ammonium chloride buffer (0.83% NH4Cl and 5
mM Tris buffer, pH. 7.2) at 37 C to lyse RBC. After washing with RPMI
1640/1% FBS, cells were suspended in culture medium (RPMI 1640
supplemented with 20 mM HEPES, 10 mM sodium pyruvate, 2 mM
L-glutamine, 50 nM 
-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. One hundred microliters of responder B6
cells were added to a U-bottom 96-well plate (Corning, Corning, NY),
mixed with the same number of gamma-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 µg/ml) was used as a positive control. One microcurie of
[3H]TdR was added to each well in the last
16–18 h of a 3-day culture. Labeled cells were harvested onto
filtermats (Skatron Instruments, Sterling, VA) with a Skatron 12-well
cell harvester. The counts per minute of the filter membrane were
measured in scintillation liquid (Cytoscint) on Beckman LS 6000IC.
For the proliferation analysis of T cell subsets, responder (B6) cells 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. The labeled cells were resuspended in culture medium and incubated with irradiated B6 (syngeneic), B/c (donor-type), or CBA (third-party) stimulator cells (2 x 106/ml). On day 4 cells were harvested and stained with anti-mouse CD3e-R-PE (clone 145-2C11), CD4-biotin (clone H129.19), and streptavidin-CyChrome (BD PharMingen). 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). Cells in lymphocyte gate, Topro 3 negative (viable cells), stained positively for CD3 were analyzed for CFSE intensities (CD3+CD4+ as CD4+; CD3+CD4- as CD8+).
CTL effector differentiation in vivo
RBC-free splenocytes were prepared as described above. One million cells were used for Ab staining in ice-cold PBSA (PBS with 1% BSA). Cells were incubated with 10 µg normal rat IgG to block Fc binding sites. After washing, the cells were stained with 0.5–1 µg anti-mouse CD8a-FITC (clone 53-6.7), CD62L-R-PE (clone MEL-14), and CD44-CyChrome (clone IM7; BD PharMingen). The stained cells were washed, and three-color flow cytometry was performed on a FACScan cytometer. Cells in lymphocyte gate stained positively for CD8a were analyzed for CD62L and CD44 expression. CTL effectors (CTLe) were identified as the CD8+CD62Llow CD44high population (23).
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 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 100 as maximal release. The specific cytolysis was calculated as % = (cpmsamples - cpmspontanous)/(cpmmax - cpmspontanous).
Statistical analysis
Comparisons were made using 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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We have shown that although B/c hearts are rejected in 7 ± 2
days (mean survival time (MST) ± SD) in naive WT B6 mice, they
are lost in an accelerated manner within 1.4 ± 0.3 days in B6
mice sensitized at day -10 with donor-type (B/c) skin grafts
(5). Although the humoral immune response is involved,
cell-mediated mechanisms play a dominant role in the accelerated
rejection cascade in this model (5). Indeed, neutrophils
contribute 50–60% of cells, with the remainder comprised of
macrophages (23%) and CD4 (14%) and CD8 (8%) T cells; NK
and B cells contribute <1% of the graft infiltrate, whereas IL-2R
expression is noted on some 15% of leukocytes. Moreover, RNase
protection assay analysis showed that accelerated rejection is
associated with intragraft mRNA expression of a number of chemokines
(and their receptors), including macrophage inflammatory protein-1
(CCR1, CCR5); monocyte chemoattractant protein-1 (CCR2); RANTES and
macrophage inflammatory protein-1 (CCR5); inducing protein-10
(CXCR3); plus the neutrophil chemokine receptor, CXCR2; and the
pan-leukocyte chemokine receptor, CXCR4. Rejection in this model is
also accompanied by intragraft expression of the key T cell-associated
cytokines, IFN-
, plus IL-6 and IL-10.
Treatment of WT hosts with anti-CD154 (MR1) mAb every second day
between the day of skin (day -10) and the day of cardiac (day 0)
engraftment prevents host sensitization and results in long term (>100
days) cardiac graft acceptance with features of transplantation
tolerance (22). To analyze whether CD154 blockade can
successfully prevent graft rejection in sensitized hosts, we
administered MR1 mAb on the day of cardiac engraftment (day 0). Unlike
in naive mice (Fig. 1
A), a
single dose of MR1 mAb was only marginally effective (MST ± SD,
4 ± 1 days) in 10-day sensitized mice (Fig. 1B),
suggesting that CD154 blockade is ineffectual in preventing rejection
in primed recipients.
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C, these long
term sensitized recipients rejected B/c hearts in an accelerated,
rather than acute, manner (MDT, 4 ± 1days), and MR1 mAb treatment
failed to affect graft survival (MST, 5 ± 1days). Similar results
were recorded with hearts transplanted up to 60 days after skin-induced
sensitization. Thus, in marked contrast to the efficacy of CD154
blockade to prevent acute rejection in naive recipients,
peri-transplant administration of anti-CD154 mAb failed to
ameliorate accelerated rejection in sensitized mice. CD154 blockade fails to affect activation of alloreactive CD8+ T cells in sensitized recipients
We have shown that CD8+ T cells are
principal effectors in the accelerated rejection response, and the main
targets of the CD154 blockade in presensitized recipients
(22). The failure to inhibit alloreactive
CD8+ T cells may represent a possible reason why
rejection still occurs despite CD154 blockade in sensitized hosts.
Thus, we contrasted the activation status of CD8+
T cells in sensitized mice with that in naive recipients. Spleen cells
harvested on day 10 post-transplant from skin-sensitized or naive
recipients of cardiac allografts treated with MR1 mAb on day 0 were
analyzed for their alloreactivity in in vitro MLR and cytotoxicity
assays. By that time, presensitized recipients had already rejected
their grafts, whereas naive recipients maintained well-functioning
transplants. As shown in Fig. 2A, splenocytes from
sensitized hosts proliferated more vigorously against donor-type
alloantigen compared with cells from naive hosts
(p < 0.0005), but comparably against
third-party Ags. FACS staining of CFSE-labeled splenocytes in bulk
cultures against alloantigens has revealed vigorous proliferation of
both CD4+ and CD8+ T cells
in sensitized hosts, but selectively diminished
CD8+ T cell proliferation in naive recipients
(Fig. 2B). However, normal CD8+ T
cells were able to proliferate if stimulated with Con A (comparable to
rejecting CD8+ T cells; data not shown),
indicating no overall CD8+ T cell defects in
naive recipients. Thus, increased MLR counts in sensitized recipients
resulted from the augmented proliferation of alloreactive
CD8+ T cells. To confirm the activation of
alloreactive CD8+ T cells, we also measured
CD8-mediated cytotoxicity after 5–6 days of in vitro culture. Indeed,
as shown in Fig. 3, stimulated cells from
sensitized, but not naive, hosts effectively lysed donor-type target
cells (75 vs 31% cytolysis, respectively, at E:T cell ratio of 50:1).
Thus, CD154 blockade fails to prevent activation of alloreactive
cytotoxic CD8+ T cells in sensitized
mice.
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Since CD154 blockade prevents allograft rejection in naive
recipients, its failure in sensitized recipients raised the question of
whether only naive, not primed, CD8+ T cells may
require CD154 costimulation for their activation. Because of limited
proliferation and differentiation of naive CD8+ T
cells in unseparated splenocytes in vitro against alloantigens, we
monitored the activation of alloreactive CD8+ T
cells using an in vivo model system developed by Mobley and Dailey
(23, 24). In this model, B6 recipient mice were stimulated
with B/c skin grafts. Indeed, as shown in Fig. 4, allogeneic, but not syngeneic, skin
grafting triggered increased expression of splenic
CD8+ T cells with
CD44highCD62Llow phenotype
on day 10 (33 vs 3.7% in naive mice). These cells were functionally
identified as activated alloreactive CD8+
cytotoxic T cells. As shown in Fig. 4, CD154 blockade in naive B6
recipients at the time of skin grafting prevented the induction of
CD8+ cytotoxic T cell subset (2.8%). The CD154
requirement for naive CD8+ CTL activation was
also confirmed in the CD154 knockout (KO) system, in which allogeneic
skin grafts failed to activate CD8+ T cells
(5.4%). Thus, CD154 costimulation signals are required for activation
of naive alloreactive CD8+ T cells.
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Primed alloreactive T cells consisted of either effector (day 10
after skin grafting) or memory (day 40 after skin grafting) cells in
sensitized recipients. To address the question of whether these primed
T cells may still require CD154 signaling when reactivated by
allostimulation, we used in vitro and in vivo systems in parallel to
monitor the activation of CD8+ T cells from
primed recipients in the absence or the presence of CD154 blockade.
Splenocytes from skin-grafted recipients (10- or 40-day sensitization
model) were first stimulated in vitro with alloantigen plus MR1 mAb for
5 days, followed by CD8+ T cell
differentiation/cytotoxicity assessment. As shown in Fig. 5A, CD154 blockade failed to
prevent activation of primed effector/memory cytotoxic T cells, as
evidenced by both differentiation of CTLe (31% of
CD62LlowCD44high cells in
total CD8+ T cells in untreated or MR1-treated
recipients) and effective lysis of target cells comparable with that of
untreated T cells (Fig. 5B; 47 and 53% cytolysis at E:T
cell ratio of 50:1 in MR1 mAb-treated and untreated recipients,
respectively). However, unlike with CD154 blockade, CTLA4Ig-mediated
blockade of the CD28 costimulation pathway effectively inhibited
activation of primed CD8+ T cells. The percentage
of CTLe decreased from 31% in untreated controls to 6.4% after
CTLA4Ig treatment (Fig. 5A), whereas cytolysis diminished
from 53% in untreated controls to 36% in CTLA4Ig-treated in vitro
culture (Fig. 5B).
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, there was a marked increase in the
number of
CD44highCD62LlowCD8 cells
triggered by the second skin graft (from 6.5 to 15.5%). CD154 blockade
at the time of the second skin graft failed to inhibit the
CD44highCD62LlowCD8
increase (15.7% in MR1 mAb treated vs 15.5% in untreated controls).
In contrast, and consistent with our in vitro data, CTLA4Ig-mediated
blockade of CD28 effectively inhibited the induction of the
CD44highCD62Llow CD8 subset
by the second skin allograft (6.4% in CTLA4-Ig treated vs 15.5% in
untreated controls). Thus, activation of primed memory
CD8+ T cells was CD154 blockade resistant.
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The presence of CD154 blockade-resistant
CD8+ T cells prompted us to design adjunctive
strategy to improve the efficacy of CD154 therapy in sensitized
recipients. As there are no definitive markers suitable for selective
depletion of memory/effector CD8+ T cells, we
transiently depleted the whole CD8+ T cell
population. Thus, B6 mice sensitized with B/c skin grafts (day -10 or
-40) were treated from day -2 for 3 consecutive days with a depleting
CD8 (2.43) mAb, and then challenged with cardiac allografts (day 0),
immediately followed by a single dose of MR1 mAb. Indeed, unlike
control groups with intact CD8 subset plus MR1 mAb treatment (MST, 3.5
days) or CD8 depletion without MR1 mAb treatment (MST, 5.5 days), only
adjunctive CD8 mAb plus CD154 mAb therapy resulted in long term graft
acceptance (>50 days; n = 6) in the 10-day
sensitization protocol (Fig. 7) and the
40-day sensitization protocol (>20 days; n = 4; not
shown). Hence, CD8 T cell depletion represents a key prerequisite for
therapeutically effective CD154 costimulation blockade in sensitized
transplant recipients. As a control, adjunctive anti-CD4 mAb (days
-2 to 0) plus anti-CD154 mAb therapy was also tested in sensitized
mice. Unlike in the anti-CD8 mAb treatment group, transient
depletion of CD4+ T cells had a marginal effect
on the efficacy of subsequent CD154 blockade in sensitized recipients,
with all cardiac allografts rejecting by 18 days (Fig. 7).
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50% from the
transient depletion (Fig. 8A).
Controls consisted of CD4 mAb- and MR1 mAb-treated mice examined 40
days after heart transplant, when CD4+ T cell
recovery was close to 80%. Interestingly, as shown in Fig. 8B, newly generated CD8+ T cells in
CD8/CD154 mAb-treated recipients bearing well-functioning grafts were
well below the control level (<3%
CD8+CD44highCD62Llow
in peripheral CD8+ T cells). In contrast,
activated alloreactive CD8+ T cells were readily
detectable in CD4/CD154 mAb-treated recipients, which did reject their
grafts 20 days earlier (24%
CD8+CD44highCD62Llow
in peripheral CD8+ T cells). As a single dose of
MR1 mAb was given 40 days before that analysis, our findings indicate
that initial CD154 blockade had a long-lasting suppressive effect on
naive alloreactive CD8+ T cell development. In
addition, CD154 blockade-resistant activation of primed
CD8+ T cells may be CD4 help independent, as
suggested by CD4 depletion controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike preventive regimens, peri-transplant administration of MR1 mAb
alone failed to affect accelerated rejection of cardiac allografts in
both skin-sensitized (days -10 and -40) models. In addition, we
detected alloreactive activated CD8+ CTLs in both
types of the primed mice despite CD154 blockade at the time of cardiac
engraftment. To explore putative mechanisms of CD154 blockade-resistant
rejection and CD8+ T cell activation, we asked
whether activation of alloreactive CD8+ T cells
may have different costimulation requirements in naive vs primed
environments. To address that issue, we first examined in vitro
CD8+ T cell responses using splenocytes from
naive or skin-sensitized B6 mice in the absence or the presence of MR1
mAb. Increased CD154 mAb-independent CD8+ T cell
proliferation and differentiation against alloantigen were readily
observed with primed T cells. Since naive CD8+ T
cells responded less vigorously in vitro, and we were not sure whether
ex vivo manipulation affects the functional state of splenocytes, we
adapted (23, 24) and modified an in vivo model system to
study alloreactive CD8+ T cell activation. Direct
measurement of recently activated CD8+ T cells in
the periphery from skin-grafted WT mice treated with anti-CD154 mAb
or from untreated CD154 KO mice revealed that CD154 signals are indeed
required for activation of naive alloreactive
CD8+ T cells. However, restimulation with the
same donor-type skin graft in primed mice augmented CTL
differentiation/cytolysis despite concomitant anti-CD154 mAb
therapy. This indicates the emergence of CD154
costimulation-independent activation of CD8+ T
cells in primed mice, representing most likely either effector or
memory CD8+ T cell generation. Spleen T cells
from these mice triggered accelerated rejection of cardiac allografts
after infusion into otherwise normal recipients despite concomitant MR1
mAb treatment, documenting that CD154 blockade-resistant
CD8+ primed/memory T cells were transferable (Y.
Zhai, unpublished observations). Interestingly, both naive and
memory/effector CD8+ T cells require CD28
costimulation signaling for their activation, which indicates the
functional hierarchy of costimulation molecules in T cell activation.
Indeed, we have previously shown the efficacy of CTLA4-Ig therapy to
prevent allograft rejection in sensitized rats (25).
Moreover, consistent with our current CD8 activation data, the effect
of CD28 blockade on cardiac allograft survival in skin-sensitized mice
(day -10 model) in our ongoing studies was superior to that of CD154
blockade (MST, 12 and 2 days, respectively).
We then attempted to address the functional importance of these costimulation blockade-resistant primed memory CD8+ T cells in the allograft rejection cascade. Although adjunctive infusion of a depleting anti-CD8 depleting mAb into already sensitized recipients eliminates all CD8+ T cells, its additional therapeutic effect relative to CD154 monotherapy was targeted mainly at the primed CD8+ T cell, because the CD154 blockade alone was sufficient to control the naive CD8+ T cell population. The prolongation of graft survival after combined peri-transplant CD8 depletion and CD154 blockade stresses the importance of primed CD8+ T cells in costimulation blockade-resistant rejection. To the best of our knowledge, this study is the first to document the differential requirement of CD154 costimulation signals for activation of naive vs primed (effector/memory) CD8+ T cells.
This study complements our recent findings on the key role of alloreactive CD8+ T cells as the prime targets of CD154-targeted therapy and effectors in the immune rejection cascade in sensitized transplant recipients (22). The issue of whether alloreactive CD8+ T cells represent the targets for CD154 blockade in transplant recipients has been controversial. It was hypothesized that CD4-dependent CD8+ T cell activation may rely on CD154 costimulation signals that activate DCs (13, 14, 15). In our case, however, alloreactive CD8+ T cell activation in vivo was both CD4 dependent and independent, as CD4+ T cell depletion or the CD4 KO environment decreased, but id not completely abolish, CD8+ T cell activation (Y. Zhai, L. Meng, R. W. Busuttil, M. H. Sayegh, and J. W. Kupiec-Weglinski, manuscript in preparation). Thus, we favor the idea that there is a CD4-independent, CD154-dependent pathway for alloreactive CD8+ T cell activation. A similar conclusion has been drawn from allogeneic murine tumor models (26, 27). In addition, it has been shown that long term expansion of adoptively transferred 2C TCR Tg CD8+ T cells in Ag-bearing F1 hosts depends on CD154 costimulation signals via CD4+ T cells (28). It has also been documented, however, that intragraft proliferation, homing, and cytokine production by Tg-TCR+ (from BM3 or DES mice) CD8+ T cells was CD154 blockade independent (29). In a multiple minor histocompatibility Ag-mismatched skin allograft model, CD154 blockade was more effective in preventing CD4-mediated than CD8-mediated rejection in WT recipients (30). It delayed CD8+ T cell-mediated rejection, resulting in significant prolongation of graft survival rather than its permanent acceptance. In this case, activation of CD8+ T cells was CD4+ T cell dependent. In an intestinal transplant model, CD154 blockade exerted some therapeutic effect in CD8 KO, but not in CD4 KO, recipients (31). Perhaps, the efficacy of CD154 blockade to target CD4 or CD8 T cell subsets may be a quantitative issue, depending on the downstream events after CD4/CD8 T cell activations in the processes of graft rejection. More importantly, CD8+ T cells do not represent a homogenous population; susceptibility or resistance to the CD154 blockade may not necessarily apply to the entire population, but rather to a specific T cell subset. Indeed, the asialo-GM1+ CD8+ T cell subset was shown to be responsible for the rejection of allogeneic skin grafts despite simultaneous CD28 and CD154 costimulation blockade (32). In addition, CD40-independent pathways of CD4+ T cell help for priming of CD8+ CTLs were demonstrated in a TCR Tg model (33).
Our results support the role of CD154 costimulation signals in activation of alloreactive CD8+ T cells, but limit its effect on the naive CD8+ T cell population. Importantly, we have determined that both effector as well as memory T cells may be potentially CD154 blockade resistant. This adds an additional level of complexity to distinct roles of T cell costimulation pathways in CD8+ T cell activation. Indeed, costimulation requirements may vary under different physiological or experimental conditions with known variables, including Ag types or doses (34, 35), the precursor frequencies of responding cells (36), other stimulating help (e.g., cytokines) (37, 38, 39), as well as functional T cell status (40).
Our finding that primed memory cells CD8+ T have differential requirements for CD154 costimulation compared with their naive counterparts is of clinical significance in improving the efficacy of current costimulation blockade-based therapeutic regimens. The priming of alloreactive CD8+ T cells occurs not only during allo-transplantation or following multiple blood transfusions, but may also result from viral or bacterial infections as well as other immune reactions due to the broad cross-reactivity of alloreactive T cells (41, 42). In the latter case, the frequency of primed T cells may not be as high as that triggered by allogeneic stimulation. However, these CD8+ T cells may well escape the CD154 blockade and then contribute to subacute or chronic-type rejection episodes. Therefore, as the CD154 blockade fails to completely control alloreactive CD8+ T cells, adjunctive targeting of CD8+ T cells to optimize therapeutic effects in transplant recipients may be warranted.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; CTLe, CTL effector; KO, knockout; MST, mean survival time; WT, wild type.
Received for publication June 4, 2002. Accepted for publication August 13, 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, Y. Wang, Z. Wu, and J. W. Kupiec-Weglinski Defective Alloreactive CD8 T Cell Function and Memory Response in Allograft Recipients in the Absence of CD4 Help J. Immunol., October 1, 2007; 179(7): 4529 - 4534. [Abstract] [Full Text] [PDF]
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 A. Valujskikh and X. C. Li Frontiers in Nephrology: T Cell Memory as a Barrier to Transplant Tolerance J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2252 - 2261. [Full Text] [PDF]
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L. K. Selin and M. A. Brehm Frontiers in Nephrology: Heterologous Immunity, T Cell Cross-Reactivity, and Alloreactivity J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2268 - 2277. [Abstract] [Full Text] [PDF]
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Z. Liu, H. Dai, N. Wan, T. Wang, S. Bertera, M. Trucco, and Z. Dai Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism J. Immunol., April 1, 2007; 178(7): 4260 - 4266. [Abstract] [Full Text] [PDF]
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H. Xu, P. M. Chilton, M. K. Tanner, Y. Huang, C. L. Schanie, M. Dy-Liacco, J. Yan, and S. T. Ildstad Humoral immunity is the dominant barrier for allogeneic bone marrow engraftment in sensitized recipients Blood, November 15, 2006; 108(10): 3611 - 3619. [Abstract] [Full Text] [PDF]
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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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D. C. Neujahr, C. Chen, X. Huang, J. F. Markmann, S. Cobbold, H. Waldmann, M. H. Sayegh, W. W. Hancock, and L. A. Turka Accelerated Memory Cell Homeostasis during T Cell Depletion and Approaches to Overcome It. J. Immunol., April 15, 2006; 176(8): 4632 - 4639. [Abstract] [Full Text] [PDF]
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B. Koehn, S. Gangappa, J. D. Miller, R. Ahmed, and C. P. Larsen Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation. J. Immunol., March 1, 2006; 176(5): 2691 - 2696. [Abstract] [Full Text] [PDF]
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M. D. Vu, M. R. Clarkson, H. Yagita, L. A. Turka, M. H. Sayegh, and X. C. Li Critical, but Conditional, Role of OX40 in Memory T Cell-Mediated Rejection J. Immunol., February 1, 2006; 176(3): 1394 - 1401. [Abstract] [Full Text] [PDF]
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I. W. Nasr, Y. Wang, G. Gao, S. Deng, L. Diggs, D. M. Rothstein, G. Tellides, F. G. Lakkis, and Z. Dai Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells J. Immunol., May 15, 2005; 174(10): 6161 - 6168. [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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 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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G. Chalasani, Q. Li, B. T. Konieczny, L. Smith-Diggs, B. Wrobel, Z. Dai, D. L. Perkins, F. K. Baddoura, and F. G. Lakkis The Allograft Defines the Type of Rejection (Acute versus Chronic) in the Face of an Established Effector Immune Response J. Immunol., June 15, 2004; 172(12): 7813 - 7820. [Abstract] [Full Text] [PDF]
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Y. Chen, P. S. Heeger, and A. Valujskikh In Vivo Helper Functions of Alloreactive Memory CD4+ T Cells Remain Intact Despite Donor-Specific Transfusion and Anti-CD40 Ligand Therapy J. Immunol., May 1, 2004; 172(9): 5456 - 5466. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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F. G. Lakkis and M. H. Sayegh Memory T Cells: A Hurdle to Immunologic Tolerance J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2402 - 2410. [Full Text] [PDF]
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M. A. Brehm, T. G. Markees, K. A. Daniels, D. L. Greiner, A. A. Rossini, and R. M. Welsh Direct Visualization of Cross-Reactive Effector and Memory Allo-Specific CD8 T Cells Generated in Response to Viral Infections J. Immunol., April 15, 2003; 170(8): 4077 - 4086. [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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