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: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions1
*
Department of Surgery, Section of General Surgery, and Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109;
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132; and
Department of Surgery, Division of Transplantation, Ohio State University School of Medicine, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient
(IFN--/-) C57BL/6 mice can rapidly reject BALB/c
cardiac allografts. When depleted of CD8+ cells, both WT
and IFN--/- mice rejected their allografts, indicating
that these mice share a common CD4-mediated, CD8-independent mechanism
of rejection. However, when depleted of CD4+ cells, WT mice
accepted their allografts, while IFN--/- recipients
rapidly rejected them. Hence, IFN--/-, but not WT mice
developed an unusual CD8-mediated, CD4-independent, mechanism of
allograft rejection. Allograft rejection in IFN--/-
mice was associated with intragraft accumulation of IL-4-producing
cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this
form of rejection was resistant to treatment with anti-CD40 ligand
(CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell
depletion studies verified that anti-CD40L treatment failed to
prevent CD8-mediated allograft rejection in IFN--/-
mice. However, anti-CD40L treatment did prevent CD4-mediated
rejection in IFN--/- mice, although grafts were
eventually rejected when CD8+ T cells repopulated the
periphery. The IL-4 production and eosinophil influx into the graft
that occurred during CD8-mediated rejection were apparently
epiphenomenal, since treatment with anti-IL-4 mAb blocked
intragraft accumulation of eosinophils, but did not interfere with
allograft rejection. These studies demonstrate that a novel,
CD8-mediated mechanism of allograft rejection, which is resistant to
experimental immunosuppression, can develop when IFN- is limiting.
An understanding of this mechanism is confounded by its association
with Th2-like immune events, which contribute unique histopathologic
features to the graft but are apparently unnecessary for the process of
allograft rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN-, the quintessential Th1 cytokine, falls into the formative, but
inessential, category. When present, IFN- can contribute
significantly to the acute rejection process by driving macrophages
(5) and endothelial cells (6) into a
proinflammatory status that favors T cell allosensitization and the
rapid development of destructive alloimmunity. Given that murine
cardiac allograft rejection is associated with prominent IFN-
production (7, 8), it appears that IFN--associated
rejection processes are sensitive to most experimental
immunosuppressants, including CTLA4Ig and mAb to CD4 and CD40 ligand
(CD40L).3
Nevertheless, mice that are genetically deficient for IFN-
(IFN--/-), can also develop rapid and
vigorous acute allograft rejection responses (9).
Curiously, these IFN--independent allograft rejection processes are
insensitive to most immunosuppressive strategies, including combination
costimulatory blockade with anti-CD40L mAb and CTLA4Ig
(10), a suppressive strategy that is powerful enough to
subvert the acute rejection of fully MHC-mismatched skin allografts
(11).
We have investigated the immunobiology of allograft rejection in
IFN--/- cardiac graft recipients. In this
report we demonstrate that IFN--/- allograft
recipients recruit several unusual immune components to the allograft
site, some of which are formative, but inessential, and others of which
are apparently irrelevant to the rejection process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type (WT) and IFN--/- female
C57BL/6 (H-2b) and BALB/c
(H-2d) mice between 6 and 12 wk of age were
obtained from The Jackson Laboratory (Bar Harbor, ME).
Medium
The culture medium used in these studies was DMEM supplemented with 0.27 mM L-asparagine, 1.4 mM L-arginine HCl, 14 µM folic acid, 5 x 10-5 M 2-ME (all obtained from Sigma, St. Louis, MO), 1.6 mM L-glutamine, 10 mM HEPES buffer, 1.0 mM sodium pyruvate, 100 U/ml penicillin/streptomycin, and 2% FCS (all obtained from Life Technologies, Grand Island, NY).
Heterotopic cardiac transplantation
WT and IFN--/- mice were transplanted
with vascularized cardiac allografts obtained from WT or
IFN--/- donors, respectively. Donor hearts
were anastomosed to the great vessels in the abdomen as described by
Corry et al. (12). In this model, the transplanted heart
is perfused with the recipient mouse’s blood and resumes contraction.
Transplant function was monitored by daily abdominal palpation. Myocyte
damage and the intensity of the graft’s infiltrate were assessed by
routine hematoxylin and eosin (H&E) staining of paraffin-embedded
sections.
Recovery of graft-infiltrating cells (GIC)
Groups of three transplanted hearts were removed, pooled, minced, and digested with 1 mg/ml collagenase A (Roche, Indianapolis, IN) for 30 min at 37°C. Tissue debris was allowed to settle at 1 x g, and the suspension containing GIC was harvested by pipette. RBC were lysed by hypotonic shock, GIC were passed through a 30-µm pore size nylon mesh, and viable leukocytes were enumerated by trypan blue exclusion. For differential enumeration, GIC were placed on slides with a cytocentrifuge and stained with Wright’s stain.
In vivo treatment with mAb
Hybridomas secreting anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) were obtained from American Type Culture Collection (Manassas, VA). The hybridoma secreting anti-CD40L (clone MR1) was provided by Randy Noelle (Dartmouth, Lebanon, NH). Anti-CD4, anti-CD8, and anti-CD40L mAb were purified and resuspended in PBS by Ligocyte Pharmaceuticals (Bozeman, MT). Purified anti-IL-4 (clone 11.B11) was provided by Craig Reynolds (National Cancer Institute, Bethesda, MD). For initial T cell subset depletion, allograft recipients were injected i.p. with 1 mg of anti-CD4, anti-CD8, or both mAb on days -2 and 0. Where indicated, cardiac allograft recipients were given an additional 1 mg of anti-CD8 mAb at 2, 4, 6, and 8 wk posttransplant to prevent repopulation of the periphery with CD8+ T cells. T cell subset depletion was verified by flow cytometry at the time of organ harvest. For inductive anti-CD40L therapy, mice were injected i.p. with 1 mg of MR1 on days 0, 1, 3, 5, and 7 posttransplant. To neutralize endogenous IL-4, allograft recipients were injected i.p. with 2 mg of anti-IL-4 mAb on days -1, 1, 3, 5, and 7.
Competitive IL-4 ELISA
A standard IL-4 ELISA (PharMingen, San Diego, CA) was modified to measure the IL-4-neutralizing capacity in the sera of allograft recipients that were treated with anti-IL-4 mAb. Briefly, rIL-4 samples (2.5 and 5 ng/ml) were incubated with PBS, dilutions of experimental sera, or purified 11.B11 (1.0, 0.1, or 0.01 ng/ml) for 30 min on ice. Samples were then assessed for serologically detectable IL-4 by ELISA (PharMingen) according to the manufacturer’s protocol. Absorbance was determined at 405 nm using an EL 800 microplate reader (Bio-Tek Instruments, Winooski, VT). Data are reported as the mean OD of triplicate samples.
Flow cytometry
Splenocytes and GIC were dual labeled with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 or FITC-conjugated anti-CD45RB and PE-conjugated anti-CD8 (all mAb obtained from PharMingen). To verify cell depletion, as opposed to cell coating by the anti-CD4 or anti-CD8 mAb used for in vivo treatment, samples were also stained with FITC-conjugated goat anti-rat IgG (Jackson ImmunoResearch, West Grove, PA). Cell analysis was performed on a Becton Dickinson FACScan (San Jose, CA) using forward vs side scatter to gate on cells.
Enzyme-linked immunospot (ELISPOT) assay for IL-4-producing cells
The ELISPOT assay used to quantify alloantigen-primed IL-4-producing cells has been described previously (13). Capture and detection anti-IL-4 mAb were obtained from PharMingen. Polyvinylidene fluoride-bottom plates (Jade Scientific, Canton, MI) were coated overnight with capture anti-IL-4 mAb (11.B11, 2 µg/ml), blocked for 90 min with 1% BSA in PBS at room temperature, and washed three times with PBS. Irradiated (2000 rad) donor splenocytes (4 x 105) were added to each well followed by 1 x 106 recipient splenocytes or 1 x 105 recipient GIC. Plates were incubated for 24 h at 37 C, then washed three times with PBS, and washed four times with PBS-Tween 20 (0.05%). Biotinylated detection anti-IL-4 mAb (BZD6–24G2, 2 µg/ml) was added to each well and incubated overnight at 4 C. Plates were washed three times with PBS-Tween 20, and HRP-conjugated strepavidin (1/2000 dilution; Dako, Carpinteria, CA) was added for 90 min at room temperature. Plates were washed four times with PBS, developed with 3-amino-9-ethylcarbazole, washed with H20, and air dried. Spots were enumerated using an ImmunoSpot Series 1 ELISPOT Analyzer (Cellular Technology, Cleveland, OH).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
The first series of studies was designed to identify the cell
types that associate with the allograft rejection process in
IFN--/- mice. Initially,
CD4+ and CD8+ T cells were
examined. Cohorts of C57BL/6 IFN--/- mice or
WT C57BL/6 mice were transplanted with
IFN--/- or WT BALB/c heterotopic cardiac
allografts, respectively, and treated with a depleting anti-CD4 mAb
(GK1.5), a depleting anti-CD8 mAb (2.43), both, or neither. Cardiac
allograft rejection was determined by cessation of palpable allograft
pulsation. As shown in Table I
, both WT
and IFN--/- mice rejected cardiac allografts
within 8 days. Treatment of WT allograft recipients with anti-CD4
mAb prolonged allograft function for >60 days, whereas treatment with
anti-CD8 mAb failed to prolong allograft function (mean graft
survival, 9 days). This suggests that CD4+ T
cells are both necessary and sufficient for allograft rejection in WT
mice, whereas CD8+ T cells are neither necessary
nor sufficient. In contrast, treatment of
IFN--/- allograft recipients with
anti-CD4 mAb had little effect on allograft survival (mean graft
survival, 11.6 days). Syngeneic grafts were accepted indefinitely in
anti-CD4-treated IFN--/- mice (data not
shown), ruling out unusual problems with cardiac transplantation or
anti-CD4 therapy in IFN--/- mice. As
with WT allograft recipients, treatment of allograft recipients with
anti-CD8 mAb only minimally prolonged allograft survival (
16
days). To achieve substantially prolonged allograft survival in
IFN--/- mice (>30 days), they must be
treated with both anti-CD4 and anti-CD8 mAbs. This indicates
that either CD4+ T cells or
CD8+ T cells can independently mediate allograft
rejection in IFN--/- mice. In contrast, only
CD4+, but not CD8+, T cells
can independently mediate rejection in WT mice.
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support the idea that IFN--/- mice employ an
unusual CD8+ T cell population that can act
independently of CD4+ T cells during the acute
rejection process. Splenocytes were obtained on day 10 from
CD4-depleted WT or IFN--/- cardiac allograft
recipients. GIC were also obtained from
IFN--/- recipients. As previously reported
(14), anti-CD4 treatment of WT recipients markedly
reduces the number of GIC, thereby limiting the availability of this
cell population for study. These leukocyte populations were dual
stained with anti-CD8 and anti-CD45RB mAb. This dual-labeling
strategy discriminates activated from resting
CD8+ T cells, since T cells exhibit reduced
expression of CD45RB subsequent to cellular activation
(15). As shown in Fig. 1, flow cytometry demonstrated that
the spleens of IFN--/- allograft recipients
exhibited a higher proportion of activated
(CD45RBlow) CD8+ T cells
than did the spleens of WT allograft recipients. Furthermore, the
majority of CD8+ T cells that accumulated within
the rejecting allografts of IFN--/- mice
expressed the activated CD45RBlow phenotype.
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). Fig. 2A
represents an H&E-stained section of a day 10 allograft harvested from
an anti-CD4-treated IFN--/- recipient.
An intense, widespread leukocytic infiltrate was noted, which upon
higher magnification (Fig. 2B) disrupted the myocardial
architecture. This infiltrate was rich in granulocytes (Fig. 2C), including both polymorphonuclear leukocytes (PMNs;
white arrows) and eosinophils (black arrows). Granulocytes are not a
common feature of unmodified rejection in WT recipients, where GIC are
primarily mononuclear in nature (16). For comparison, Fig. 2D depicts the histology of a day 10 allograft harvested
from an anti-CD4-treated WT recipient. Note the mild, diffuse
infiltrate and the preservation of myocardial architecture. The few GIC
that could be recovered on day 10 were mononuclear cells (Fig. 2D, inset, x1000).
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, the prototypic Th1 cytokine, it is
reasonable to expect compensatory production of IL-4, the prototypic
Th2 cytokine, during an immune response (17, 18).
Furthermore, the presence of eosinophils within rejecting allografts
has been associated with the production of Th2 cytokines (16, 19), and Th2 have been implicated in graft rejection (reviewed
in Ref. 20). Hence, we asked whether IL-4-producing Th2
were a prominent feature of allograft rejection in
IFN--/- recipients. To this end, splenocytes
obtained 10 days posttransplant from anti-CD4-treated WT or
IFN--/- allograft recipients were stimulated
in ELISPOT cultures with irradiated donor strain splenocytes. GIC
obtained from IFN--/- recipients were also
included in this analysis. As shown in Fig. 3, splenocytes and GIC obtained from
IFN--/- allograft recipients contained
substantial numbers of IL-4-producing Th2. In contrast, splenocytes
obtained from anti-CD4-treated WT allograft recipients displayed
negligible IL-4 production. As in Fig. 1
, GIC were not obtained in
sufficient number from WT recipients for ELISPOT analysis.
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-/- recipients treated with anti-CD4
mAb accumulated several unusual cell types relative to their WT
counterparts: 1) activated CD8+ T cells, 2)
IL-4-producing leukocytes (presumably CD8+ T
cells), 3) PMNs, and 4) eosinophils. Furthermore, immunologic evidence
indicated that the CD8+ T cells were directly
involved in the acute rejection process in
IFN--/- mice, in that depletion of both
CD4+ and CD8+ T cells
resulted in prolonged graft survival (Table I). This is further
supported by the finding that adoptive transfer of these
CD8+ T cells into IFN--sufficient SCID
allograft recipients resulted in acute allograft rejection that was
associated with a similar histologic picture as that seen in
IFN--/- recipients (data not shown).
Contribution of CD40-CD40L interactions to allograft rejection in
IFN--/- mice
Previous studies by others suggested that allograft rejection in
IFN--/- mice is insensitive to treatment
with anti-CD40L mAbs (10). We confirmed this
observation in Table II, which
demonstrates that WT recipients retain the allografts for >60 days if
treated with anti-CD40L mAb. In contrast,
IFN--/- mice rapidly rejected their
allografts within about 12 days despite anti-CD40L mAb treatment.
Our preceding studies (Table I) demonstrated that allograft rejection
in IFN--/- mice could be independently
mediated by either CD4+ or
CD8+ T cells. This suggests that at least one of
these T cell subpopulations must be insensitive to the
immunosuppressive effects of anti-CD40L. To test this hypothesis,
IFN--/- mice were depleted of either
CD4+ or CD8+ T cells,
engrafted with BALB/c hearts, and then treated with inductive
anti-CD40L mAb to determine whether this treatment could subvert
either the isolated CD4-mediated or CD8-mediated allograft rejection
process. When the rejection process was mediated by
CD8+ T cells (i.e., initial treatment with
anti-CD4 mAb), allograft rejection was not influenced by
anti-CD40L therapy. In contrast, when the rejection process was
mediated by CD4+ T cells (i.e., initial treatment
with anti-CD8 mAb), the rejection process was delayed until about
45 days. This was approximately the period of time required for
CD8+ T cells to repopulate the periphery
following the initial depletion with anti-CD8 mAb (see Fig. 4). Indeed, if these mice were
repetitively treated with anti-CD8 mAb to prevent repopulation with
CD8+ T cells (anti-CD8 five times), the
allografts survived longer (>64 days). These observations illustrate
several important points: 1) the CD4+ T cells of
IFN--/- mice are, in fact, sensitive to anti-CD40L
therapy, as they are in WT mice; 2) the resistance to the
suppressive effects of anti-CD40L therapy in
IFN--/- mice is due to their utilization of
an unusual, anti-CD40L mAb-insensitive, CD4-independent,
CD8+ T cell population to reject the allograft;
and 3) the mechanisms by which anti-CD40L therapy subverts
CD4-mediated rejection cannot protect the graft against the assault of
the CD8-mediated rejection process, even if the
CD4+ T cell responses are allowed to develop
first.
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-/- recipients that were
initially treated with anti-CD8 and anti-CD40L mAb was
associated with the development of Th2 responses.
IFN--/- allograft recipients were given
inductive anti-CD40L mAb and either a single peritransplant dose of
anti-CD8 mAb (anti-CD8 once) or repetitive doses of
anti-CD8 mAb (anti-CD8 five times) to forestall repopulation by
CD8+ T cells. Fig. 4 (left
panel) illustrates that CD8+ T cells
were readily detectable by flow cytometry by day 47 posttransplant in
the spleens of IFN--/- allograft recipients
given a single dose of anti-CD8 mAb. Furthermore, these spleens
contained large numbers of donor alloantigen-reactive, IL-4-secreting
cells that were detectable by ELISPOT. The allografts of these mice
were rejected (Fig. 5) and contained an
aggressive infiltrate (Fig. 5A) rich in PMNs and eosinophils
(Fig. 5B). Hence, the return of CD8+
cells following initial depletion was associated with graft rejection
and the onset of Th2-associated events.
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-/- allograft recipients that were
given inductive anti-CD40L and repetitive doses of anti-CD8
mAb, CD8+ cells were not readily detectable on
day 64 when the experiment was terminated (Fig. 4, right
panel). Furthermore, these spleens contained very few
IL-4-producing cells. The allografts in these mice continued to
function normally and were virtually free of leukocytic infiltration
(Fig. 5C).
Role of IL-4 in allograft rejection in IFN--/-
mice
The accumulation of IL-4-producing cells and eosinophils
(21) within the rejecting allografts of
IFN--/- mice suggested that IL-4 and related
phenomena might contribute to rejection (22, 23). To test
this possibility, IFN--/- allograft
recipients were depleted of CD4+ T cells and
treated with the neutralizing anti-IL-4 mAb, 11.B11. Others have
used this approach to effectively reverse the biologic effects of IL-4
in vivo using a variety of experimental systems (21, 24, 25, 26, 27). A total of 10 mg of 11.B11 was administered to
allograft recipients, since this dose has been proven to reverse in
vivo the biologic effects of IL-4 (25, 26, 27). Interestingly,
treatment with anti-IL-4 mAb did not prolong graft survival (Fig. 6A), indicating that IL-4 was
not an integral component of the allograft rejection mechanism employed
by CD8+ T cells in
IFN--/- mice. To verify that mice treated
with our 11.B11 regimen did, in fact, have adequate IL-4-neutralizing
capacity available, a competitive ELISA was established to measure the
amount of anti-IL-4 activity in the sera of these mice (Fig. 6B). Indeed, a 1/10,000 dilution of sera obtained at the
time of rejection from 11.B11-treated mice was as effective at
neutralizing 5 ng/ml rIL-4 as 1 ng/ml purified 11.B11 in this assay.
Hence, our 10-mg treatment with 11.B11 mAb provided mice with
substantial IL-4-neutralizing capacity. Furthermore, neutralizing
endogenous IL-4 with 11.B11 had a major impact on the nature of the
infiltrate that accumulated in rejecting allografts (Fig. 6C), thus illustrating a biologic effect of this therapy. As
has been reported in tumor systems (21), neutralizing IL-4
abrogated the influx of eosinophils into the allografts, but had no
apparent effect on lymphocyte or monocyte recruitment. Interestingly,
the number of PMNs was increased by anti-IL-4 treatment, perhaps
reflecting compensation for the loss of eosinophil recruitment.
Collectively, these observations suggest that IL-4 production and
downstream immunologic effects such as eosinophilia are not requisite
components of the CD8-mediated allograft rejection process that
develops in IFN--/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated immune activation, and CTL-mediated allograft
destruction. Hence, the transplant community was generally surprised to
learn that rapid allograft rejection continues to occur in mice that
are genetically deficient for IL-2 (28), IFN-
(9), IL-12 (29), and perforin
(30). Obviously, many backup mechanisms exist that can
compensate for the loss of selected immune components, and the concept
of one prototypic mechanism of acute allograft rejection is
unrealistic. The current immunologic challenge is to appreciate the
nature of these alternative response mechanisms and the rules for their
selection and deployment.
Our studies on acute allograft rejection in the
IFN--/- mouse provide a particularly good
example of alternative pathway utilization by the immune system.
IFN- is a pluripotent proinflammatory mediator that so dominates the
tone and character of an immune response that responses involving
IFN- production are collectively known as Th1 responses, one of two
fundamental organizational patterns of T cell reactivity (17, 18). IL-4 production represents the opposing pattern of T cell
reactivity, and responses that are dominated by IL-4 have been termed
Th2 responses. An enormous literature documents that acute allograft
rejection is commonly associated with IFN- production and all of its
downstream consequences. Thus, acute allograft rejection is commonly
considered to be a Th1 response. Furthermore, it has been proposed that
preferential induction of graft-reactive Th2 may be protective in the
context of transplantation due to the inhibitory effects of Th2
cytokines on Th1 function. However, an emerging body of evidence
indicates that Th2 may also be involved in graft rejection
(20). Given that IFN- is an integral element of acute
allograft rejection, it is intriguing that
IFN--/- mice efficiently reject experimental
allografts (9). In the absence of IFN- the development
of IL-4-producing Th2 is favored (17, 18), and
Th2-associated downstream events such as eosinophilia may mediate
tissue damage (21). Hence, it seemed reasonable to propose
that acute rejection in IFN--/- mice may be
mediated by IL-4-producing Th2, thus redirecting the choice of
rejection mechanisms to one which included eosinophils (16, 19).
This study challenges this hypothesis and demonstrates that the acute
cardiac allograft rejection process in
IFN--/- mice is fundamentally different from
the rejection process that develops in WT mice. These differences
include the presence of donor alloantigen-reactive, IL-4-producing T
cells in the spleens of the allograft recipients and within the
rejecting allografts themselves (Fig. 3). Induction of this Th2
response was associated with a prominent intragraft accumulation of
PMNs and eosinophils (Figs. 2, 5, and 6C). None of these
events occurs in unmodified WT allograft recipients (16)
or WT recipients that have been depleted of CD4+
T cells (14) (Figs. 2 and 3). Interestingly, serologic
neutralization of IL-4 in IFN--/- allograft
recipients did not interfere with the development of acute allograft
rejection (Fig. 6A), although it effectively abrogated
intragraft eosinophilia (Fig. 6C). At the very least, this
indicates that the IFN--/- recipient
threshold for IL-4-associated acute rejection is below that for
IL-4-associated intragraft eosinophilia. More likely, these studies
indicate that IL-4 production and intragraft eosinophilia are not
necessary for the acute allograft rejection in
IFN--/- mice and may occur only as
irrelevant epiphenomena. These observations may, in fact, counter the
argument that IFN--/- mice are predisposed
to an alternative Th2 mechanism of acute allograft rejection. Indeed,
these observations underscore the fact that the appearance of various
immune components during an immune response merely reflects their local
availability and not their essential participation.
Unlike the rejection process in WT mice, cardiac allograft rejection in
IFN--/- mice is insensitive to
immunosuppression with anti-CD4 (Table I) and anti-CD40L (Table II) mAb. Others have observed a similar insensitivity allograft
rejection in IFN--/- mice to costimulatory
blockade with combination CTLA4Ig and anti-CD40L mAb therapy
(10). Hence, the rejection mechanism in
IFN--/- mice apparently does not require
costimulation through either the CD28/B7 or the CD40/CD154 pathway.
This report further documents these findings. In addition, this study
revealed that cardiac allograft rejection in
IFN--/- mice does not require
CD4+ T cell help (Table I). Hence, an unusual
CD8-mediated, CD4-independent mechanism of acute allograft rejection
develops in IFN--/- mice. It should be noted
that this is not unique to the C57BL/6
IFN--/- mouse strain, in that BALB/c
IFN--/- recipients of C57BL/6 allografts
reject their transplants in a similar fashion (D. K. Bishop,
unpublished observations). We have also observed that a similar
CD8-mediated, CD4-independent mechanism of rejection is used when
allogeneic hepatocytes are implanted into WT C57BL/6 mice
(31). Others have reported similar observations in the
rejection of intestinal allografts (32), the development
of autoimmune diabetes (33), tumor rejection
(34), skin graft rejection (35), and
hapten-induced contact hypersensitivity (36). Hence, this
CD8-mediated effector mechanism may be used in a wider variety of
immunologic settings than has been previously appreciated.
The mechanisms by which CD8+ T cells mediate
acute allograft rejection in IFN--/- mice
remains to be determined. The obvious hypothesis is that the
CD8+ T cells directly mediate lytic destruction
of graft cells subsequent to TCR mediated recognition of allogeneic MHC
class I molecules on graft tissues. However, freshly isolated GIC
obtained from IFN--/- recipients depleted of
CD4+ cells mediate only minimal lytic activity in
standard 4-h 51Cr release assays, and the number
of precursor CTL in the spleens of these mice is not increased, as
determined by limiting dilution analysis (data not shown). This
contrasts the Th1-dominated rejection process in unmodified WT mice,
where GIC mediate appreciable lytic activity (37), and the
spleens contain large numbers of precursor CTL (14).
Alternatively, the CD8+ T cells indirectly
mediate tissue destruction by recruiting or activating other
destructive mechanisms mediated by macrophages or PMNs, which are
present in significant numbers in IFN--/-
allografts (Fig. 6). Indeed, a recent report links PMNs with the
deleterious Th2 response in experimental leishmaniasis
(38). Fig. 6 of this study also demonstrates that the
large numbers of eosinophils that accumulate in these grafts are not
necessary for rejection. However, it is not known whether this
eosinophil component contributes to the rejection process. Although not
directly demonstrated, our studies suggest that donor-reactive
CD8+ T cells are the source of IL-4 in
IFN--/- allograft recipients. Donor-reactive
IL-4-producing cells were present in the spleens and grafts of
anti-CD4-treated IFN--/- allograft
recipients (Fig. 3), and these IL-4-producing cells reappeared as
CD8+ cells repopulated the periphery following an
initial treatment with anti-CD8 mAb (Fig. 4). In vitro,
CD8+ T cells can be induced to make IL-4 in a
CD4+ T cell-independent manner (39).
However, the IL-4 neutralization studies depicted in Fig. 6 indicate
that the CD8+ effector function may not be
dependent upon endogenous IL-4 production. It is interesting to note
that IL-4 causes CD8+ T cells to switch from
perforin-mediated lysis to Fas-mediated lysis (40). Thus,
IL-4 may have some formative, albeit not essential, effects in
CD8-mediated allograft rejection.
It is not clear why IFN--/- recipients of
cardiac allografts develop the CD8-dependent mechanism of cardiac
allograft rejection. Perhaps the development of CD8-dependent rejection
is actively restrained in WT mice by the presence of IFN-. This
hypothesis needs to be tested. Alternatively, the use of the
CD8-dependent rejection mechanism by IFN--/-
mice might result from an unusual pattern of immunologic hardwiring
that develops during ontogeny in the absence of IFN-. The latter
possibility is intriguing because of its implications for clinical
transplantation. Studies by Hutchinson et al. (41) have
demonstrated that the capacity to produce specific cytokines is
genetically controlled and highly variable in the human. In their
studies, 25% of the tested population expressed a genotype that
encodes deficient IFN- production (42). Our studies
suggest that these individuals may develop compensatory immune
capacities that change their immunologic approach to allografts and
perhaps their susceptibility to certain immunosuppressants.
In contrast, the IFN--/- recipients of
cardiac allografts develop CD4+ T cell responses
that appear quite similar to those that develop in WT mice.
Specifically, the CD4+ T cells can independently
mediate allograft rejection in IFN--/- mice,
and this rejection is susceptible to therapy with anti-CD4 (Table I) or anti-CD40L (Table II) mAbs. In unrelated studies we have
found that treatment of murine cardiac allograft recipients with
gallium nitrate (43), anti-CD4 mAb (43),
or anti-CD40L mAb (C. G. Orosz, unpublished observations) does
not merely block the development of allosensitization and acute
rejection, but permits the emergence of CD4+
regulatory T cells that actively prohibit donor-reactive cell mediated
immunity via the production of TGF-
and IL-10 (44). The
question remains of whether CD4+ regulator T
cells would emerge in immunosuppressed
IFN--/- allograft recipients when
CD8+ T cells are removed. If so, the generation
of the regulator T cells responsible for allograft acceptance is
IFN- independent. Others have suggested that IFN- is needed to
facilitate the induction of allograft acceptance (10).
Furthermore, we have observed that IFN--/-
mice treated with anti-CD40L only during the peritransplant period
will accept cardiac allografts indefinitely, provided that they are
repetitively depleted of CD8+ T cells (Table II).
This has two important implications. First, it supports the possibility
that regulatory T cells may emerge in the absence of IFN-. Second,
it suggests that these regulatory T cells cannot interfere with the
activation and function of the CD8+ mediators of
allograft rejection. Indeed, the re-emergence of
CD8+ T cells in anti-CD40L-treated,
CD8-depleted IFN--/- allograft recipients,
which occurs 40 days following the initial depletion (Fig. 4),
precipitates the rapid induction of acute rejection (Table II,
Fig. 5).
The existence of this difficult to control, CD8-mediated mechanism of acute rejection has several serious implications. First, it jeopardizes experimental efforts to induce allograft tolerance solely through the generation of donor-reactive, regulatory CD4+ T cells. It now becomes important to determine what conditions foster the development of these immunosuppression- and regulation-insensitive CD8+ mediators of allograft rejection and whether they are susceptible to either endogenous mechanisms of immune regulation or exogenous agents of immunosuppression. Finally, it is important to determine whether and when this unusual CD8-mediated mechanism of allograft rejection develops in humans and primates.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. Keith Bishop, Transplant Immunology Research, Section of General Surgery, A560 MSRB II, Box 0654, University of Michigan Medical Center, Ann Arbor, MI 48109.
3 Abbreviations used in this paper: CD40L, CD40 ligand; GIC, graft-infiltrating cells; H&E, hematoxylin and eosin; IFN-/-, IFN- deficient; PMN, polymorphonuclear leukocyte; WT, wild type; ELISPOT, enzyme-linked immunospot.
Received for publication October 11, 2000. Accepted for publication December 18, 2000.
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