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Prepublished online as a Blood First Edition Paper on May 15, 2003; DOI 10.1182/blood-2003-02-0586.
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Blood, 1 September 2003, Vol. 102, No. 5, pp. 1920-1926
TRANSPLANTATION
Mechanisms of donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation
Sergio A. Quezada,
Bruce Fuller,
Lamis Z. Jarvinen,
Mercedes Gonzalez,
Bruce R. Blazar,
Alexander Y. Rudensky,
Terry B. Strom, and
Randolph J. Noelle
From the Department of Microbiology & Immunology, Dartmouth Medical
School, Lebanon, NH; the Division of Bone Marrow Transplantation, University
of Minnesota, Minneapolis, MN; the Howard Hughes Institute, University of
Washington, Seattle, WA; and the Beth Israel Deaconess Medical Center,
Department of Medicine, Harvard, Boston, MA.
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Abstract
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Induction of transplantation tolerance to alloantigens without general
immunosuppression remains an enduring challenge. Injecting a donor-specific
transfusion (DST) of spleen cells together with blocking  CD154 antibody
prior to graft transplantation is an effective way to induce long-lived graft
acceptance. Using a novel T-cell receptor (TCR) transgenic (Tg) model of
CD4+ T-cell–mediated rejection, this study sheds new insights
into the cellular basis for enhanced graft survival induced by DST and
CD154. The study shows that DST and CD154 induce an early,
robust, abortive expansion of the Tg T cells that results in profound anergy.
This is contrasted with the more delayed, regional, productive response
elicited by an allogeneic graft. Studies show that the induction of tolerance
to the allograft induced by DST is mediated by indirect presentation by host
antigen-presenting cells. Based on these observations, we conclude that DST
and CD154 preemptively tolerize the alloreactive T-cell compartment to
prohibit subsequent responses to the immunogenic allograft.
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Introduction
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For more than 2 decades, it has been recognized that the infusion of whole
blood from donors (donor-specific transfusion [DST]) into recipients can
prolong allograft survival in humans, as reviewed in
humans1 and
mice.2 More
recently, it has been shown that the prolonged survival of allografts induced
by DST is synergistically enhanced by the coadministration of CD154
(CD40L).3 In
some cases, permanent graft survival of allogeneic grafts can be observed with
CD154 and DST. While some insights into the underlying mechanisms of
graft survival have been gained, our understanding of the cellular basis for
allograft tolerance is incomplete. A number of recent studies using this means
to induce graft survival have shown that active suppression plays an essential
role in silencing the effector function of graft-rejecting T cells. While a
role of regulatory T cells (Treg's) in this process has been tentatively
identified, little is known about the fate and function of alloreactive
effector T cells upon exposure to DST and CD154.
It is hypothesized that one cellular mechanism of DST/CD154-induced
graft tolerance is the inactivation of alloreactive CD4+ and
CD8+ effector T
cells.2 Based on
studies of Buhlmann et
al,4 it was
speculated that direct recognition of the infused allogeneic B cells (DST) by
alloreactive T cells in the CD154-suppressed environment resulted in
inadequate DST activation, with reduced up-regulation of costimulatory
molecules, and cytokine production by the DST. Under these conditions, it was
hypothesized that clonal tolerance of the alloreactive T-cell population was
rendered. Whether this tolerance was the result of clonal ignorance, anergy,
or apoptosis has not been resolved because of the inherent constraints of the
systems used. The use of TCR transgenic (Tg) models to study the basis for
T-cell tolerance has permitted great insights into the spectrum of possible
defects that can account for the tolerant state. Alloreactive T-cell fate and
function have been directly assessed in the studies presented herein through
the use of a novel CD4+ TCR Tg model wherein alloantigens expressed
by the DST, together with CD154 blockade, induce profound
unresponsiveness. As such, questions as to the fate and function of normal and
tolerant alloreactive T cells have been addressed.
Many hypotheses of DST-induced tolerance are based on the proposition that
host alloreactive T cells directly recognize alloantigen on the DST. However,
recent studies have shed doubt on this premise. Using a spectrum of DST
allotypes, matched or mismatched with the host, studies by Niimi et
al5 suggested that
presentation of alloantigen-derived peptides in the context of
self–major histocompatibility complex (MHC) was essential for the
beneficial effect of haplotype-shared blood transfusions. If this is true,
then processing and presentation of DST-derived allopeptides by the host
antigen-presenting cells (APCs) is critical in alloantigen-induced tolerance.
Accepting this proposition, one must presume that the synergy observed with
CD154 is due to its impact on the host APC machinery. Blockade of CD154
exerts profound effects on the function, longevity, and differentiation of
dendritic cells
(DCs).6 As such, the
effect of CD154 blockade is to shorten the duration of antigen presentation by
the DCs and to limit their capacity to be immunogenic. Preventing DC
maturation, and at the same time delivering DST, may be a superlative means
for inducing tolerance to alloantigen-derived epitopes that are presented by
host DCs. It is thought that autologous apoptotic cells that are processed and
presented by nonmatured host DCs are critical to maintain peripheral
tolerance.7-9
DST and CD154 may, in fact, take advantage of similar mechanisms to
those used in maintaining peripheral self-tolerance in order to induce
allotolerance. Data presented herein directly address and measure the
contribution of indirect presentation to DST-induced graft survival.
Using the CD4+ TCR Tg system (TEa), in which receptor
specificity is directed to an allopeptide derived from I-E and
presented in the context of class II MHC (H-2b), we have tracked
the behavior of CD4+ alloreactive T cells in response to DST,
CD154, and an allograft. Data show that DST induces a rapid, robust
expansion of alloreactive T cells that is abortive and results in profound
T-cell anergy. Blocking of CD154 does not qualitatively change the response to
DST, but serves to magnify the intensity of the unresponsiveness. In contrast
to the alloresponse to DST, the Tg T-cell response to the allograft is
regional, robust, and productive, giving rise to highly responsive
alloreactive T cells that ultimately infiltrate the graft and mediate
rejection. Therefore, the preemptive and near complete unresponsiveness
induced by DST/CD154 serves to extinguish the subsequent response to
the allograft. Furthermore, the data clearly show that DST does not directly
present alloantigen to the host, and serves only as an alloantigen depot,
providing allopeptides to be presented by host APCs. In light of these
findings, a novel and cohesive model of DST/CD154-induced allotolerance
is presented.
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Materials and methods
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Mice
C57BL/6 MHC class II–deficient mice and recombination activating gene
(RAG) knock-out (KO) mice were purchased from Taconic (Germantown, NY).
CB6F1 mice (hybrid of C57BL/6 and Balb/c), C57BL/6, C57BL/6 CD45.1,
and Balb/c mice were purchased from the National Cancer Institute (Frederick,
MD). The TEa CD4+ TCR Tg
mice10 were kindly
provided by Dr Alexander D. Rudensky (University of Washington, Seattle).
CD4+ Tg cells express a TCR that recognizes the peptide
ASFEAQGLANIAVDKA in the context of I-Ab. This peptide corresponds
to the positions 52 to 68 from the -chain of I-E class II molecules and
is expressed in all APCs from H-2b/I-E+ strains
(CB6F1). C57BL/6 mice are H-2b but
I-E–, whereas BALB/c are H-2d and I-E+,
and therefore their F1 hybrids are H-2b/I-E+
and able to directly present antigen. The TEa Tg mice were bred to C57BL/6,
congenic C57BL/6 CD45.1 (Ly5.2) mice at the animal facility at Dartmouth
Medical School. All mice were bred and housed in microisolator cages in a
pathogen-free facility.
Skin grafting
Skin grafting was performed as a modification of the technique used by
Markees et al.11
Briefly, age-matched male CB6F1 mice were used as donors of both
spleen cells (DST) and skin grafts. In some groups, age-matched C57BL/6 grafts
were used as negative control skin donors. CD45.1 and CD45.2 were used as
additional congenic markers for tracking the TEa Tg T cells. Age-matched
CD45.1+ TEa Tg mice were used as donors of alloreactive T cells.
Quantification of the TEa Tg T cells was done by staining with
anti–V2-TCR–phycoerythrin (PE) and
anti–CD4–fluorescein isothiocyanate (FITC). Recipients were
age-matched RAG KO mice. On day –1, donor CB6F1 mice were
killed and tail-skin grafts (0.5 cm x 0.5
cm)12 or
mechanically disaggregated spleen cell suspensions were prepared from them.
Recipient mice were injected with or without 5 x 107 DST
cells and 1 x 106 TEa Tg T cells in 500 µL Hanks balanced
salt solution by tail vein injection (intravenously) and 500 µg of
CD154 (clone MR-1) or control hamster immunoglobulin (H-Ig) in
phosphate-buffered saline (PBS) intraperitoneally. Mice were injected with
CD154 or H-Ig 3 times per week for the duration of the experiment. On
day 0, recipient mice were anesthetized with 50 µg per gram body weight of
each of ketamine and xylazine injected intraperitoneally (15 mg/mL in PBS),
and CB6F1 or C57BL/6 skin grafts were prepared using established
methods.12
Rejection was defined as the day on which less than 20% of the skin graft
remained.
All antibodies were obtained from Pharmingen (San Diego, CA).
Statistical analysis
Survival data were analyzed using the Kaplan-Meier method with the Wilcoxon
rank test and the log-rank test used to verify the significance of the
difference in survival between groups. P values less than .05 were
considered statistically significant.
Fluorescence-activated cell analysis
Lymph node (LN) cell suspensions were stained to analyze expansion and
purity of TEa Tg T cells in all different groups. This was assessed by
staining with anti–CD45.1-FITC and anti–V2-TCR-PE. All
antibodies were obtained from Pharmingen.
Purification and adoptive transfer of in vivo–stimulated TEa Tg
T cells
Age-matched RAG KO mice were injected intravenously with 5 x
107 DST cells and 1 x 106 CD45.1+ TEa
Tg T cells, and intraperitoneally with 500 µg of either CD154 or
H-Ig 3 times per week. On day 7, recipients or naive TEa mice were killed, and
their LNs were harvested and mechanically disaggregated. Cells were positively
selected for CD45.1 using anti–CD45.1-biotin and streptavidin magnetic
beads according to the manufacturer's instructions (Miltenyi Biotech, Auburn,
CA), stained with anti–V2-TCR-PE and anti–CD45.1-FITC to
determine percentage of selected T cells (recipients, > 70%; naive TEa,
> 90%), and counted. Age-matched male or female RAG KO recipients were
injected with 1 x 105 TEa Tg T cells harvested from
DST+/– CD154–treated RAG
KO mice. Skin grafting of CB6F1 grafts was performed, and grafts
were monitored as described in "Skin grafting." Antibodies were
obtained from Pharmingen.
In vivo expansion of TEa Tg T cells
On day 0, recipient RAG KO mice received 1 x 106
CD45.1+ TEa Tg T cells and either (1) 5 x 107 DST
cells (from CB6F1 or Balb/c donors) with or without CD154,
(2) a CB6F1 skin graft with or without CD154, (3) 5 x
107 DST cells (CB6F1 or Balb/c) and a CB6F1
skin graft with or without CD154, or (4) a C57BL/6 skin graft. Mice
treated with CD154 received 500 µg 3 times per week, as described in
"Skin grafting," and were compared with mice receiving the same
dose of H-Ig. Mice were killed either at day 7 for in vitro recall assays or
at day 9 for determination of in vivo expansion. For comparison of local
versus nonlocal expansion, lymph node cells were harvested, and mechanically
disaggregated. Total number of cells per lymph node was determined and
standardized to the percentage of TEa Tg–positive cells determined by
flow cytometry.
In vitro recall responses
Media used were RPMI (Bio-Whitaker, Walkersville, MD) containing 10% fetal
bovine serum, 2 mM L-glutamine, 5 x
10–5 M 2-mercaptoethanol, 100 U/mL penicillin, and
100 µg/mL streptomycin. Splenocytes from CB6F1 or C57BL/6 mice
were irradiated with 30 Gy (3000 rad), and 5 x 105 cells per
well were added to separate wells of a 96-well plate in 100 µL media. At
day 7, LN cells from the different groups were harvested and selected for
CD45.1 expression as described in "Purification and adoptive transfer of
in vivo–stimulated TEa Tg T cells," stained with
anti–CD45.1-FITC and anti–V2-TCR-PE, counted, and plated in
96-well plates. TEa Tg T cells (20 000) in 100 µL media were added to wells
containing 100 µL irradiated CB6F1 cells, 100 µL irradiated
C57BL/6 cells, or 100 µL media. Some wells containing irradiated
CB6F1 cells or irradiated C57BL/6 cells received 100 µL media
and no TEa Tg T cells. Replicate plates were incubated to assess cytokines and
proliferation. Proliferation and cytokines were assessed as previously
described.13
Assessment of cell proliferation by measuring cytoplasmic dye
dilution
To follow in vivo kinetics of division, TEa Tg T cells were labeled with
the intracellular fluorescent dye 5- (and 6-) carboxyfluorescein diacetate
succinimidyl ester (CFSE) obtained from Molecular Probes (Eugene, OR) prior to
adoptive transfer into naive RAG KO recipients. At days 4 and 8, local and
nonlocal LN cells were recovered and assayed by multicolor flow cytometry,
gating in the TEa Tg cell population (as previously described) to detect the
dilution of the dye caused by cell proliferation. Each successive cellular
generation exhibits half of the intensity of CFSE fluorescence of its parental
population.
Assessment of graft infiltration
On day 0, recipient RAG KO mice received 1 x 106
CD45.1+ TEa Tg T cells and either (1) 5 x 107 DST
cells with or without CD154, (2) a CB6F1 skin graft with or
without CD154, (3) 5 x 107 DST cells and a
CB6F1 skin graft with or without CD154, or (4) a C57BL/6
skin graft. Mice treated with CD154 received 500 µg 3 times per
week, as described throughout "Materials and methods," and were
compared with mice receiving the same dose of H-Ig. On days 4, 7, and 14 skin
grafts were removed, formalin fixed, and processed for hematoxylin and eosin
(H&E) staining by established protocols.
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Results
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Skin graft rejection and prolonged graft acceptance induced by DST
and CD154 blockade in a CD4+ TCR transgenic model
Initial experiments demonstrated that TEa Tg T cells could mount an
effective graft rejection response in vivo. As shown in
Figure 1, transfer of
CD4+ TEa Tg T cells into
RAG–/– C57BL/6 mice alone
mediates graft rejection with similar kinetics to those seen in non-Tg
rejection systems (mean survival time [MST] = 18.6 days in the Tg system; MST
= 10 days in the non-Tg
system).12 To
evaluate if treatment with CD154 with or without DST could interfere
with TEa Tg T-cell–mediated graft rejection, recipient
RAG–/– mice were injected with or
without 5 x 107 CB6F1 spleen cells and 500 µg
CD154 or control H-Ig. One day after injection, treated mice received a
CB6F1 skin graft. CD154 or control antibody was subsequently
administered 3 times per week. Treatment with DST + CD154 significantly
delayed graft rejection by the CD4+ TEa Tg T cells (MST = 72.4
days, P < .0001), and the degree to which it delayed the rejection
is also similar to that seen in polyclonal systems (MST = 51
days).12 DST or
CD154 alone significantly delayed graft rejection (MST = 35.5 days,
P < .05; MST = 41.7 days, P < .0002, respectively),
but not to as great a degree as with DST and CD154 together (MST = 72.4
days, P < .0001). All groups treated with CD154 or DST have
some rare long-term surviving grafts (> 100 days), the largest percentage
of these being in the DST + CD154–treated group (DST = 13%,
CD154 = 11%, DST + CD154 = 24% long-term surviving grafts).
Control groups received TEa Tg T cells and a syngeneic graft or no TEa Tg T
cells and CB6F1 grafts with or without DST. None of these groups
ever rejects its grafts (data not shown).
CD154 reduces the in vivo expansion of alloreactive Tg T cells
induced by DST or an allogeneic skin graft in vivo
The in vivo response profile of the TEa Tg T cells in response to DST,
CD154, and/or a skin allograft was measured. Briefly, on day 0, mice
received 1 x 106 TEa Tg T cells and either (1) 5 x
107 DST cells with or without CD154, (2) an allogeneic
CB6F1 skin graft with or without CD154, (3) 5 x
107 DST cells and a CB6F1 skin graft with or without
CD154, or (4) a syngeneic C57BL/6 skin graft. CD154 or H-Ig was
administered 3 times per week. As shown in
Figure 2, at day 9,
CB6F1 skin grafts induced extensive expansion of TEa Tg T cells in
draining LNs compared with expansion induced in nondraining lymph nodes. On
the other hand, DST induced systemic expansion of TEa Tg T cells with
equivalent numbers in all LNs. In all groups, treatment with CD154
blocked TEa Tg T-cell expansion by approximately 50%.
CD154 accentuates the alloreactive T-cell unresponsiveness
induced by DST
The combined administration of CD154 and DST enhanced graft survival
more effectively than either agent alone. To address the cellular basis for
enhanced survival, the functional activity of TEa Tg T cells following in vivo
tolerization was measured. Tg T cells were purified from treated and untreated
mice and restimulated in vivo and in vitro. Briefly, CD45.1+ TEa Tg
T cells were adoptively transferred into
RAG–/– recipients together with
DST, CB6F1 skin, and with or without CD154 treatment. On day
7, mice were killed, LN cells were harvested, and the TEa Tg T cells were
purified by positive selection for CD45.1 expression. For in vivo
restimulation, equivalent numbers of "tolerized" (DST +
CD154–exposed TEa Tg T cells [1 x 105, > 70%
pure]) or control (DST + H-Ig–exposed TEa Tg T cells) TEa Tg T cells
were transferred into naive
RAG–/– recipients. A third group
of mice received only naive TEa Tg T cells (1 x 105 cells). A
CB6F1 skin graft was placed on each mouse, and rejection was
followed over time. As shown in Figure
3A, TEa Tg T cells exposed to DST + CD154 showed a reduced
capacity to reject CB6F1 skin grafts (MST = 94.75 days; P
< .0001), compared with naive controls (MST = 25.8 days). TEa Tg T cells
exposed to DST + hamster Ig (H-Ig) also showed a reduced capacity to reject
CB6F1 skin but to a less significant degree (MST = 41.11 days;
P < .0001). Thus, DST and CD154 treatment induced
substantial T-cell unresponsiveness to alloantigen restimulation in vivo.
Seeking an explanation to the hyporesponsiveness seen in
Figure 3A, we assessed the in
vitro functional responsiveness of purified TEa Tg T cells. TEa Tg T cells
were purified from treated mice (as described in "Materials and
methods"), and their proliferative and cytokine production profiles in
response to alloantigen were determined. All results presented used purified
TEa Tg T cells and represent responses on a per cell basis. As shown
in Figure 3B-D, TEa Tg T cells
from mice that were grafted with allogeneic skin had markedly higher in vitro
proliferative and cytokine production responses (interleukin-2 [IL-2] and
interferon  [IFN]) than all other groups. DST, while inducing
substantial in vivo expansion (Figure
2), did not appear to prime the TEa Tg T cells to respond upon
recall to alloantigen in vitro. Thus, alloantigen provided by allogeneic skin
compared with that provided by allogeneic spleen cells produced markedly
distinct effects on the recall response of the TEa Tg T cells in vitro. Most
interestingly, administration of DST to mice that received skin grafts
markedly reduced the recall responses to challenge with alloantigen in vitro.
Even more, concomitant administration of CD154/DST led to a more
profound inhibition of recall responses. The recall proliferative response to
allogeneic skin (65 000 cpm/culture) was reduced to less than 5% (2000
cpm/culture) by the coadministration of CD154/DST. Similarly, cytokine
production profiles in response to allogeneic skin were greatly reduced by the
coadministration of CD154/DST. In addition, tolerized TEa Tg T cells
were incapable of producing the Th2 cytokine IL-4 or to suppress skin
rejection by naive TEa Tg T cells (data not shown). Finally, no overt
phenotypic differences in cell surface profiles (CD25, CD44, and CD62L) were
apparent between the tolerized and immune Tg T cells. In summary, on a per
cell basis, DST and, moreover, CD154/DST induced profound T-cell
anergy.
DST induces an early, abortive T-cell response that preempts the
productive response to an allograft
The data thus far indicated that heightened TEa Tg T-cell recall responses
were induced by an allograft and that the coadministration of DST prior to an
allograft could block allograft-induced T-cell "priming."
Furthermore, the T-cell unresponsiveness elicited by DST was further
accentuated by CD154. One way in which DST could exert a dominant
impact over priming by an allograft was if the Tg T cells were anergized by
the DST prior to encountering the immunogenic allograft. The tempo of the
response of TEa Tg T cells to DST versus an allograft was studied in vivo by
evaluating the extent of in vivo Tg T-cell division over time. This was
achieved by following the dilution of the intracellular fluorescent dye, CFSE.
CFSE-labeled Tg TEa T cells were adoptively transferred into
RAG–/– recipients together with
CB6F1 DST, CB6F1 skin, or syngeneic skin. At days 4 and
8, cells from draining and nondraining LNs were harvested and CFSE dilution
was analyzed by flow cytometry. Figure
4 shows that at day 4, all the TEa Tg T cells in draining or
nondraining LNs from the recipient that received DST had divided, with no
remaining cells within the high-staining population. The same effect was
observed in the presence of DST and CD154, but with a reduction in
overall expansion (data not shown). At the same time point, allogeneic skin
induced limited division of the Tg cells in the draining LNs and no division
in nondraining LNs. This is demonstrated by a small number of total TEa Tg T
cells with reduced dye dilution, yet with a sizable high-staining population
still present on day 4. However, after 8 days, skin alone was able to strongly
stimulate T-cell division in draining LNs with some evident migration of
dividing cells to the nondraining LNs. As a control, syngeneic skin did not
induce TEa Tg T-cell division in draining or nondraining LNs. Therefore, DST
induces an early, systemic expansion of alloreactive T cells leading to T-cell
anergy, which may preempt the ensuing productive response to the
allograft.
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Figure 4.. The allogeneic response of TEa Tg T cells to DST and skin is temporally
and spatially separated. CFSE-labeled Tg TEa T cells were adoptively
transferred into RAG–/– recipients together
with CB6F1 DST, CB6F1 skin, or syngeneic skin as a
negative control for proliferation. At days 4 and 8, cells from draining and
nondraining LNs were harvested and CFSE dilution of the TEa Tg T cells was
analyzed by flow cytometry. Total number of TEa Tg T cells/LNs is shown for
each group.
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DST-induced hyporesponsiveness and increased graft survival is
mediated by indirect presentation of alloantigen and CD154 blockade
While the prevailing hypothesis proposes that DST-induced tolerance is due
to direct presentation of donor alloantigens to host alloreactive T cells,
this has not been rigorously tested. We evaluated the potential contribution
of direct versus indirect antigen presentation to T-cell hyporesponsiveness.
Cells from Balb/c mice (H-2d) provide the antigen (I-E), but
not the appropriate MHC-restricting element (I-Ab). Thus Balb/c DST
cannot be directly recognized by the TEa Tg T cells. If Balb/c DST results in
reduced TEa Tg T-cell response to in vitro restimulation or enhanced graft
survival in B6 RAG–/– recipients,
it can be due only to the fact that the Balb/c-derived I-E was
presented indirectly by host (H-2b) APCs. As shown in
Figure 5A, the response to an
F1 skin graft was reduced equivalently by either Balb/c or
CB6F1 DST+/– CD154.
In concordance with this observation, BALB/c, like CB6F1 DST, was
also able to induce enhanced graft survival with CD154 treatment
(Figure 5B-C). Thus, in the
absence of direct presentation, indirect presentation of alloantigen is
capable of inducing Tg TEa systemic expansion (data not shown),
unresponsiveness to in vitro restimulation, and enhanced allograft
survival.
DST is incapable of inducing TEa Tg T cell systemic expansion via
direct presentation of alloantigen
While the aforementioned data demonstrated that indirect presentation of
alloantigen was sufficient for Tg T-cell tolerance induction
(Figure 5), it did not address
the potential contribution of direct alloantigen presentation by DST. To
evaluate the contribution of direct presentation, the responsiveness of TEa Tg
T cells to CB6F1 DST in class
II–/– mice was determined. In
contrast to RAG–/– recipients,
the class II–/– recipients are
unable to indirectly present the E peptide provided by the
F1 DST. As shown in Figure
6, while F1 DST induced vigorous expansion of Tg cells
in RAG–/– mice, in the class
II–/– mice there was no expansion
of TEa Tg T cells in response to CB6F1 DST. These data strongly
indicate that the CB6F1 DST is not directly "seen" by
the TEa Tg T cells but provides allopeptides for host APC presentation.
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Figure 6.. Direct presentation of alloantigens in class II KO recipients is unable
to induce systemic expansion of Tg TEa cells. TEa Tg T cells (1 x
106) were adoptively transferred into C57BL/6
RAG–/– or class II KO recipients (n = 3 per
group) in the presence of CB6F1 skin alone or together with
CB6F1 DST. After 7 days in vivo, lymph nodes were collected and the
total number of Tg TEa cells per LN were quantified as previously
described.
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Reduced graft infiltration by TEa Tg T cells by DST and CD154
treatment
To critically evaluate the impact of CD154/DST on T-cell
infiltration and pathology, skin grafts were histologically evaluated. Skin
grafts were removed from the mice on days 4, 7, and 14, and examined by
H&E staining for infiltration and inflammation of the grafts. As can be
seen in Figure 7, mice
receiving a syngeneic graft have no inflammation or infiltration of the graft
until day 14, at which point there is very minor spongiosis of the stratum
basale of the epidermis and hair follicles, and a very minor lymphocytic
infiltrate. This is most likely due to the normal process of wound healing, as
these grafts are never rejected. In mice receiving a CB6F1 graft,
on day 4 the grafts appear the same as those seen in mice receiving syngeneic
grafts, but by day 7 there is well-established interface dermatitis. The
interface dermatitis consists of vacuolar changes in the stratum basale of the
epidermis, necrotic keratinocytes, and lymphocytic infiltrates apparent in the
dermis. By day 14, the grafts on these mice are necrosing. Mice receiving a
CB6F1 graft and DST cells show a similar, although not quite as
pronounced, pattern of graft infiltration and necrosis as seen in mice
receiving CB6F1 grafts alone. Mice receiving a CB6F1
graft together with CD154 treatment show a different pattern, with no
inflammation on day 7 with the exception of rare dead keratinocytes (typically
one dead cell per section). By day 14, the grafts on these mice are necrosing.
Skin graft sections from mice receiving a CB6F1 graft together with
DST/CD154 treatment demonstrate a few scattered dead keratinocytes on
days 4 and 7. On day 14, one can observe the beginnings of an interface
reaction, milder than that seen on day 7 of the untreated or DST-treated mice.
Thus CD154 treatment delays skin graft infiltration and together with
DST treatment can greatly delay infiltration and prolong graft survival.
Further characterization of the infiltrating lymphocytes by confocal
microscopy showed clear staining of CD4+CD45.1+ donor
TEa Tg T cells correlating to the levels of lymphocyte infiltration observed
with H&E staining (data not shown).
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Discussion
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Insights into the behavior of the CD4+ alloreactive T-cell
compartment in graft rejection and acceptance have been hampered by the lack
of the appropriate in vivo systems to visualize T-cell responses. Here we
present a novel model using alloreactive CD4+ TCR Tg T cells to
follow the fate and function of T cells specific to a major donor antigen, and
at the same time define the contribution of direct and indirect pathways of
antigen presentation to the tolerogenic process. The validity of this model
for studying graft rejection and tolerance is shown by the fact that
CD4+ T cells from a TCR transgenic mouse (TEa) demonstrate the
capacity to reject allogeneic (H-2bxd) but not syngeneic skin at a
tempo consistent with that observed with polyclonal, alloreactive T-cell
populations. Furthermore, we show that immune intervention (CD154/DST)
can enhance allograft longevity. Unique to this system is the ability to
directly measure, on a per cell basis, the response profiles of alloreactive T
cells that have been immunized or "tolerized" in vivo. Compared
with naive TEa Tg T cells, TEa Tg T cells that have been primed to an
allograft in vivo have higher proliferative indices and higher levels of
cytokine production on a per cell basis. Other studies of in vivo–primed
TCR Tg T cells have also shown that primed or memory CD4+ TCR Tg T
cells are
hyperresponsive.14
Using this system, we were able to address the cellular basis of the synergy
between CD154 and DST. First, expansion of alloreactive Tg T cells
induced by the allograft in the regional nodes is reduced by 60% by
CD154. Hence, the total number of allospecific T cells in the host is
reduced. Second, while DST alone induces profound anergy, the anergy induced
by DST/CD154 is more pronounced. That is, the proliferative
responsiveness of the residual T cells following DST/CD154 is about 30%
of that observed with DST alone (on a per cell basis). Furthermore, when
equivalent numbers of DST-treated versus DST/CD154-treated TEa Tg T
cells are transferred into naive mice
(Figure 3A), the latter retain
grafts for more than 50 days longer. Thus, the intrinsic functional capacity
of the DST/CD154-treated T cells is impaired compared with DST-treated
TEa Tg T cells. Remarkable is the fact that the major qualitative difference
between tolerized and nontolerized TEa Tg T cells resides in their capacity to
proliferate and produce proinflammatory cytokines upon restimulation;
meanwhile other parameters, such as their activation phenotype, remain
unchanged.
DST/CD154 induces a profound systemic T-cell expansion that is rapid
and results in the generation of a population of anergic T cells. The finding
that indirect presentation mediates DST-induced T-cell anergy alters the
prevailing paradigm and strongly suggests that DST-induced tolerance may use
mechanisms usually operative in inducing cross-tolerance to
self-antigens.15 In
models of peripheral T-cell tolerance to self-antigens, it has been shown that
specific subsets of host, immature dendritic
cells16-18
constitutively present self-antigens to autoreactive T cells resulting in
T-cell anergy or
deletion.15,19
It is also known that tissue destruction and apoptotic cells are superb
sources of self-antigen for delivery to these immature
APCs.7,20-22
We would propose that shortly after infusion, cells of the DST apoptose and
efficiently deliver alloantigen to host APCs for the indirect presentation of
donor allopeptides. The overwhelming tolerogenic impact of alloantigen
delivered by DST can be seen by the anergy induced by DST alone. The
synergistic actions of CD154 with DST can be explained by this same
model. First, CD154 is likely proapoptotic for the infused leukocytes,
as we know that CD154 expressed by the host would lead to activation and
longevity of the donor-derived B cells and DCs in the DST. Second, and more
important, blocking CD154 blocks the potential activation of those immature
DCs that are presenting newly acquired alloantigen from the
DST.6 It has been
shown that triggering via CD40 activates DCs and "breaks"
peripheral tolerance, and thus in the context of DST, it is important to block
endogenous CD154
function.23,24
The contrast in scope and tempo of T-cell responsiveness to DST versus the
allograft helps to explain the effectiveness of DST-induced tolerance. Studies
have shown that the timing of DST administration relative to allografting is
an important parameter in long-lived graft survival. Longer periods of time
between DST (within limits) and grafting, and multiple administrations of DST
prior to grafting can enhance graft
survival.25 All of
these parameters likely manifest as more effective measures to preemptively
induce anergy in the alloreactive T-cell compartment prior to transferring the
immunogenic allograft. The second feature of DST is its systemic impact on
alloreactive T-cell responses. As would be predicted, DST administered
intravenously systemically anergizes the alloreactive T-cell pool. In
contrast, the predominant impact of the allograft is regional, eliciting
profound local expansion and T-cell activities.
It is becoming increasingly clear that Treg's play a central role in
long-lived graft acceptance induced by
DST/CD154.26-28
However, in this Tg system, there is a lack of CD4+
CD25+ regulatory T cells, allowing us to study the independent
impact of DST/CD154 on the effector T-cell compartment. In the absence
of Treg's, it is shown that DST and CD154 induce hyporesponsiveness of
the TEa Tg T effector T cells. The hyporesponsive TEa Tg T cells when
cotransferred with naive TEa Tg T cells do not "suppress" graft
rejection, establishing that they are not regulatory in nature (data not
shown). We have similarly shown this effect of DST and anti-CD154 using
purified polyclonal CD4+CD25– T cells transferred
into RAG–/– mice (data not
shown). Future studies using this transgenic system will investigate the
dynamic interactions between regulatory and effector allogeneic T cells in
response to the tolerogenic stimuli provided by DST/CD154 therapy.
Finally, this novel TCR Tg system offers unique insights into the processes
governing immunity and tolerance in graft acceptance. While only CD4 responses
were studied in this report, the systematic inclusion of CD4+
regulatory T cells and CD8 Tg T-cell populations will allow a more expansive
understanding of how these individual alloreactive populations influence the
development of graft tolerance. The capacity to track each of these
populations independently in vivo will provide a more comprehensive
appreciation for how complex interactions between distinct alloreactive T-cell
populations result in graft rejection or acceptance.
|
Acknowledgements
|
|---|
We thank Anne Perry, MD, for interpretation of H&E sections, Evan F.
Lind for help with histology, Kathy Bennett for assistance with animal care,
and the Englert Cell Analysis Laboratory.
|
Footnotes
|
|---|
Submitted February 21, 2003;
accepted May 8, 2003.
Prepublished online as Blood First Edition Paper, May 15, 2003;
DOI 10.1182/blood-2003-02-0586.
Supported by grant A148667 (R.J.N.), CA91436 (R.J.N.), AI 34495 (B.R.B.),
2R37 HL56067 (B.R.B.), HL63452 (B.R.B.), and the Rosaline Borison fellowship
(S.A.Q.).
S.A.Q. and B.F. contributed equally to the work presented in this
study.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked "advertisement" in accordance with 18 U.S.C. section
1734.
Reprints: R. J. Noelle, Department of Microbiology & Immunology,
Dartmouth Medical School, 1 Medical Center Dr, Lebanon, NH 03756; e-mail
rjn{at}dartmouth.edu.
|
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Top
Abstract
Introduction
represents H-Ig [n = 13]; 
, DST [n = 16]; 
, 
, DST +
