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,§
*
Department of Medicine, Cleveland Veteran’s Affairs Medical Center, Cleveland, OH 44106;
Case Western Reserve University, Cleveland, OH 44106;
Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106; and
§
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2. The role of type 2 cytokines (IL-4 and
IL-5) remains controversial, as is whether alloreactive
CD4+ and CD8+ T cells behave similarly when
exposed to type 2 cytokine-enhancing manipulations. We studied the
characteristics of alloreactive CD4+ and CD8+ T
cells before and after type 2 immune deviation induced by IL-4 plus
anti-IFN- Ab. Alloreactive T cells from naive mice were low in
frequency, produced only IL-2, and were predominantly CD4+,
while alloreactive T cells from allograft-primed mice were high in
frequency, produced IFN-, IL-2, and IL-4, and were predominantly
CD8+. Type 2 immune deviation of allospecific
CD4+ T cells resulted in IL-4 and IL-5 production without
IFN-, consistent with unipolar type 2 immunity. These T cells
mediated delayed-type hypersensitivity, but not cytotoxicity. Under
identical type 2 cytokine-inducing conditions, allospecific
CD8+ T cells were primed to become IL-4, IL-5, and IFN-
producers, and exhibited cytotoxicity, but not classic delayed-type
hypersensitivity. Adoptive transfer of either cell population into SCID
recipients of allogeneic skin resulted in graft rejection, with stable
allospecific type 2 cytokine production in vivo. Adoptive transfer of
the IL-4/IL-5-producing CD4+ T cells, but not the
CD8+ T cells, induced a distinct histopathology
characterized by marked eosinophilic infiltration of the skin. We
conclude that type 2 immune deviation has differential effects on
CD4+ and CD8+ T cells and results in emergence
of alternate effector mechanisms capable of destroying
allografts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 (1, 2). These cytokines
contribute to graft destruction in a number of ways, including
up-regulating MHC II (3), costimulatory molecule (4, 5, 6), and chemokine
expression in the graft (7, 8); providing help for induction of
allograft-specific cytotoxicity (1, 2); activating macrophages and
macrophage-mediated effector mechanisms within the graft (9, 10, 11, 12, 13); and
inducing Ig class switching to complement-fixing IgG2a Abs (14). In contrast, allospecific secretion of the type 2 cytokines IL-4 and IL-5 has been associated with graft tolerance in several experimental models, including induction of neonatal allograft tolerance (15) and induction of graft tolerance using CTLA4-Ig (16) or anti-CD4 Abs (17, 18, 19). Although an allospecific immune response develops under these situations, the noninflammatory nature of the response has been hypothesized to be nondestructive as well as to prevent the emergence of pathogenic type 1 immunity (2, 16).
It is well established, however, that type 2 immunity is not truly
benign and can mediate pathogenic immune responses under a variety of
conditions. For example, IL-4 and IL-5 are chemoattractants and
activators of eosinophils that, in turn, can mediate tissue destruction
(20), in part via secretion of cationic proteins such as major basic
protein (MBP)3 (21, 22). In
addition, the initial descriptions of IL-4 and IL-5 revealed that these
so-called noninflammatory cytokines can prime allospecific
CD8+ T cells to become CTLs (23, 24, 25, 26). These experiments,
among others, provide evidence that induced allospecific type 2
immunity may indeed be destructive, not protective, to an allograft. In
support of this, both IFN- knockout (27) and IL-2 knockout mice can
reject allografts (28), and detection of type 2 cytokines has been
associated occasionally with graft rejection (29). Moreover, direct
attempts at type 2 immune deviation have not led to graft tolerance and
in some cases have resulted in accelerated rejection (30, 31, 32).
The mechanisms of allograft destruction following induction of type 2 cytokine-mediated alloimmune responses are poorly understood, however. Furthermore, it has largely been assumed that type 2 immune deviation would have similar effects on both CD4+ and CD8+ T cell subsets. As transplant rejection involves a high-frequency immune response comprised of both CD4+ and CD8+ T cells with multiple antigenic specificities, varying activation thresholds, and differing costimulatory requirements (20, 33, 34, 35, 36), it is not at all clear that this would be true. In addition, an inability to adequately measure and quantify cytokines produced by freshly isolated alloreactive CD4+ and CD8+ T cells has prevented a careful analysis of these issues.
To provide further insight into the effects of type 2 immune deviation on the cytokine profiles and functional characteristics of alloreactive T cells, our laboratory has developed a highly sensitive cytokine ELISA spot assay (33, 37). The secreted cytokines can be detected over short time intervals (<24 h in culture), thus providing an improved reflection of the in vivo immune response when compared with standard techniques (33, 37). We used this assay to evaluate cytokines secreted by alloreactive CD4+ and CD8+ T cells during allogeneic skin graft rejection and to determine the effects of IL-4-induced type 2 immune deviation on the cytokine profiles of these T cell subsets. We further evaluated the functional characteristics of the type 2 immune-deviated cell populations through analysis of cytotoxicity, ability to mediate delayed-type hypersensitivity (DTH), and ability to induce skin graft rejection. Our findings revealed that alloreactive CD4+ and CD8+ T cells respond differently to the same type 2 cytokine-inducing stimulus. The experiments also revealed that both type 2 cytokine-producing CD4+ and CD8+ T cells are capable of mediating allograft rejection, although the two cell types most likely function through different mechanisms. These results underscore the plasticity of the alloimmune response, and have important implications for future attempts at tolerance induction through immune deviation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c (H-2d), BALB/c SCID (H-2d), SJL (H-2s), C57BL/6 (B6, H-2b), and B10.Br (H-2k) mice, age 6 to 8 wk, were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in specific pathogen-free animal facilities at Cleveland Veteran’s Affairs Medical Center and Case Western Reserve University (Cleveland, OH).
Placement of skin grafts
Full thickness trunk skin allografts were placed using standard techniques (33, 37). Skin was harvested from euthanized donor mice, the s.c. fat was removed, and the skin was cut into 0.5-cm2 pieces and placed in sterile PBS until used for transplantation (less than 30 min). Recipient mice were anesthetized with pentobarbital (50 µg/g body weight) and shaved around the chest and abdomen. The skin allograft was placed in a slightly larger graft bed prepared over the chest of the recipient and secured using Vaseline-impregnated gauze and a bandage. Bandages were removed on day 7, and the grafts were then visually scored daily for evidence of rejection. The allograft was considered fully rejected when it was >90% necrotic. In selected animals, allograft rejection was confirmed histologically. All skin graft placements performed on SCID recipients were done under fully sterile conditions in a laminar flow hood. Adoptive transfers of T cells into SCID recipients were performed by i.v. injection into the tail vein.
T cell and T cell subset isolation
Splenic and lymph node T cells were isolated using commercially available murine T cell isolation columns from R&D Systems (Minneapolis, MN) following the instructions supplied by the manufacturer. Resultant cells were washed in HBSS medium, counted by trypan blue exclusion, and resuspended at appropriate concentrations for use in the various assays. An aliquot of cells was set aside for surface staining and FACS analysis.
Staining of cell surface molecules and FACS analysis
Phycoerythrin (PE)-conjugated anti-CD3 (2C11), PE-conjugated anti-CD8a, FITC-conjugated anti-CD4, FITC-conjugated anti-trinitrophenol (isotype-matched control), and PE-conjugated anti-trinitrophenol (isotype-matched control) were purchased from PharMingen (San Diego, CA). Splenocytes or T cells were labeled by direct (anti-CD3) staining, as previously described (38, 39). After three washes in PBS, the cells were fixed in fresh 1% paraformaldehyde and stored at 4°C in the dark until analyzed (within 24 h). Analysis was performed using a Becton Dickinson FACScan and accompanying software using 5000 ungated cells.
Preparation of stimulator cells
Splenic stimulator cells were prepared by incubation with mitomycin C (Boehringer Mannheim, Indianapolis, IN) at 50 µg/ml in PBS for 20 min at 37°C, followed by three washes in HBSS. The cells were counted by trypan blue exclusion and diluted for use in the various assays.
Type 2 immune deviation in vitro
Freshly isolated and purified T cells or T cell subsets were
mixed with stimulator cells, 500 U/ml IL-4 (Boehringer Mannheim), and
XMG1.2 anti-IFN- Ab (10 µg/ml, produced in our laboratory from
a hybridoma) in 2 ml of complete RPMI 1640 in 24-well plates for
5 days. The resultant cells were washed in HBSS and studied in recall
ELISA spot assays or FACS analysis.
ELISA spot assays
Ninety-six-well ELISA spot plates (Autoimmun Diagnostika,
Columbia, MD) were coated overnight with the capture Abs in sterile
PBS. R46A2, 4 µg/ml (isolated and purified from hybridoma), was for
IFN-; JES6-1A12, 3 µg/ml (PharMingen, San Diego, CA), was used for
IL-2; 11B11, 2 µg/ml (isolated and purified from hybridoma), was used
for IL-4; and TRFK5, 5 µg/ml (isolated and purified from
hybridoma), was used for IL-5. The plates were blocked for 1 h
with sterile PBS/1% BSA and washed three times with sterile PBS.
Various dilutions of splenocytes, lymph node cells, or purified T cells
(0.25–8 x 105/ml) in 200 µl of HL-1 medium
(BioWhittaker, Walkersville, MD) were placed in each well with 6
x 105 mitomycin C-treated stimulator cells (in duplicate)
and incubated at 37°C for 24 h in 5% CO2. Pilot
studies testing 1–10 x 105 mitomycin C-treated
stimulator cells demonstrated optimal detection of cytokine spots at
this concentration (not shown). Further pilot studies revealed a linear
relationship between the number of responder cells plated and the
number of detected spots for each cytokine (not shown). After washing
with PBS followed by PBS/0.025% Tween (PBST), detection Abs were added
overnight. XMG1.2 horseradish peroxidase (produced in our laboratory)
was used for IFN-, rat anti-mouse IL-4-biotin (BVD6-24G2;
PharMingen) was used for IL-4, rat anti-mouse IL-2-biotin
(JES6-5H4; PharMingen) was used for IL-2, and biotinylated TRFK4
(PharMingen) was used for IL-5. The plates were then washed three times
in PBST. Streptavidin horseradish peroxidase (Dako, Carpenteria, CA)
was added at 1/2000 dilution in PBST as a third reagent for IL-2, IL-4,
and IL-5 for 2 h, followed by three washes in PBS. The plates were
developed using 800 µl 3-amino-9-ethyl-carbazole (Pierce,
Rockford, IL; 10 mg dissolved in 1 ml dimethylformamide) mixed in 24 ml
0.1 M sodium acetate, pH 5, plus 12 µl H202.
The resulting spots were counted on a computer-assisted ELISA spot
image analyzer (T spot Image Analyzer; Autoimmun Diagnostika), which is
designed to detect ELISA spots using predetermined criteria based on
size, shape, and colorimetric density.
Cytotoxicity
Cytotoxicity was performed as published (38, 40). B6 or third-party SJL target cells were made by incubating 6 x 106 splenocytes with 2 µg/ml Con A (Sigma, St. Louis, MO) in 2 ml HL-1 medium (Biowhittaker, Walkersville, MD) for 48 h. [3H]Thymidine, 10 µCi, was added for the final 6 h. After three washes in HBSS medium, the target cells were counted and 10,000 cells were placed in each well of a round-bottom 96-well plate. In some experiments, B6-derived, MHC II-expressing, macrophage IC-21 cells (obtained from American Type Culture Collection, Manassas, VA) were used as targets. IC-21 cells were grown in six-well plates in DMEM/10% FCS, labeled with 10 µCi [3H]thymidine overnight, washed three times in HBSS, and plated at a final concentration of 10,000 cells/well. Responder CD4+ or CD8+ T cells (produced as outlined above) were added to the targets at various E:T ratios and incubated at 37°C, 5% CO2 for 5 h. The plates were harvested and counted by liquid scintillation, and the percentage of cytotoxicity was calculated as described (40).
Delayed-type hypersensitivity
DTH was performed as previously described (38, 39). Briefly, 1–2 x 106 in vitro activated CD4+ or CD8+ T cells plus 1 x 106 mitomycin C-treated, allospecific, or third-party stimulator cells in a total volume of 25 µl of PBS were injected into the ears of three naive BALB/c mice. Ear thickness was measured at 24 and 48 h with an engineer’s micrometer by an investigator blinded to the experimental groups, and the results were expressed as a difference in mean values of the thickness between the ears at time zero and 24 or 48 h.
Microscopy
Skin graft samples or ear samples were obtained at the time of sacrifice, fixed in 10% buffered Formalin, embedded in paraffin, and processed for routine histopathology. Slides were examined in a blinded fashion by a dermatopathologist (A.C.G.) and photographed.
Tissue staining for MBP
For detection of eosinophils and extracellular MBP in tissue, we utilized rabbit polyclonal antisera to murine MBP, which was prepared by Dr. Kirsten Larsen (41), and kindly provided by Dr. Gerald Gleich (Mayo Clinic, Rochester, MN). The specificity of the Ab for murine MBP and eosinophils has been previously published (41, 42). Anti-MBP was diluted 1/1000 in 1% FCS in 0.05 M Tris-buffered saline, and incubated with 5-µm paraffin sections at room temperature for 2 h. Biotinylated goat anti-rabbit Ig (Dako, Carpenteria, CA) diluted 1/200 in 1% FCS in 0.05 M Tris-buffered saline was then added for 30 min, and alkaline phosphatase-conjugated streptavidin (BioGenex, San Ramon, CA) was added for an additional 30 min. Vector Red Substrate containing 12 mg Levamisole (Sigma) was added, and sections were counterstained using modified Harris’ hematoxylin (Richard-Allen, Kalamazoo, MI).
Statistical analysis
Statistical comparisons were performed using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, we evaluated the cytokines produced by
alloreactive splenic T cells obtained from naive BALB/c mice. The
purified T cells were approximately 68% CD4+ and 32%
CD8+ by FACS (not shown). Cytokine-producing alloreactivity
was determined using an ELISA spot assay that allows detection of
cytokines produced over short time intervals (
24 h). T cells
responding to fully MHC-disparate B6 stimulator cells were low in
frequency (
40 per million) and produced predominantly IL-2 in this
24-h assay (Fig. 1
A). Previous
experiments have confirmed that these responding T cells lie within the
L-selectinhigh (CD62Lhigh) population,
suggesting that they are naive, not memory T cells (33).
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, B
and C). The purity of each isolated cell fraction was >95%
by FACS. As shown previously (33), the responses detected in assays
using these T cell preparations represent direct recognition of
allopeptides expressed in the context of allo-MHC (43). We and others
have also shown that T cells responding to allopeptides expressed in
the context of self (recipient)-MHC (indirect recognition) represent a
significant minority of the overall alloresponse and express similar
cytokine profiles to direct pathway-responsive T cells (37, 43, 44, 45, 46, 47).
ELISA spot analysis of the purified CD4+ and
CD8+ T cells confirmed a predominance of IL-2 producers in
response to alloantigenic stimulation and further revealed that
essentially all of the cytokine-producing cells derived from the
CD4+, and not from the CD8+, subfraction (Fig. 1, B and C).
To confirm the viability of the cells and to determine the effect of in
vitro priming on the alloreactive T cell population, the
CD4+ and CD8+ T cells isolated from naive
BALB/c mice were next cultured with B6 splenic stimulator cells in
vitro, and recall immune responses were determined by ELISA spot
analysis on day 5 (Fig. 1, D–F). In vitro priming of
CD8+ T cells yielded a predominance of IFN- producers
(600 spots per 106 cells plated) with 2- to 15-fold
fewer IL-2 (90 spots per 106 cells plated), IL-4 (225
spots per 106 cells plated), or IL-5 producers (35 spots
per 106 cells plated, Fig. 1F). In contrast, in
vitro priming of CD4+ T cells yielded a higher frequency of
allospecific IFN- (900 spots per 106 cells plated),
IL-2 (1400 spots per 106 cells plated), IL-4 (850
spots per 106 cells plated), and IL-5 producers (150
spots per 106 cells plated, Fig. 1E).
Interestingly, in vitro priming of unfractionated T cells resulted in
an overwhelming predominance of IFN- producers (5000 spots per
106 cells plated), although IL-2, IL-4, and IL-5 were
detected at lower frequencies as well, suggesting that the coculture of
CD4+ and CD8+ T cells had a synergistic effect
on the priming of IFN--producing cells (Fig. 1D). In sum,
although the primary alloresponse initiates with a low frequency of
IL-2-producing CD4+ T cells, both alloreactive
CD4+ and CD8+ T cells from naive mice can be
primed in short-term culture to produce a mixed type 1/type 2 cytokine
profile (and not a pure type 1 profile).
Alloreactive T cells from allograft-primed mice produce a mixed type 1/type 2 cytokine profile that derives from both CD4+ and CD8+ subfractions
To study the cytokine profiles of in vivo primed cells, we next
purified T cells and T cell subsets from the draining lymph nodes of
BALB/c mice at the time of rejection of B6 trunk skin allografts (day
11–12). Draining lymph nodes contained 58% CD4+ and 21%
CD8+ T cells by FACS in this strain combination (Fig. 2A). Allospecific cytokine
production by total lymph node cells revealed a high frequency of
IFN- (3000 spots per 106 cells plated), IL-2 (1000
spots per 106 cells plated), and IL-4 producers (1100
spots per 106 cells plated) with a few IL-5 producers
(50 spots per 106 cells plated, Fig. 2B).
Importantly, however, the high frequency and mixed profile of
cytokine-producing cells are similar to the in vitro primed
alloresponse (Fig. 1D), but contrast markedly with the low
frequency, IL-2-dominated profile found in T cells obtained from naive
mice tested in a primary recall response (Fig. 1A).
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, C and D). Interestingly, both the
CD4+ and the CD8+ fractions contained IL-2 and
IL-4 producers, but the CD8+ subfraction was the dominant
cytokine-producing population at this time point and notably contained
all of the IFN- producers. CD8+ T cells were also the
dominant cytokine producers in B6 recipients of BALB/c skin grafts,
confirming that this was not a strain-specific phenomenon (not shown). Effect of type 2 immune deviation on the cytokine profile of alloreactive T cells
Because much interest has developed in understanding the role of
type 2 cytokines in graft rejection, we next studied how type 2 immune
deviation affected the development of allospecific T cell cytokine
profiles. Previous work has implicated IL-4 as a strong inducer of type
2 immune cytokines, and IFN- and IL-12 as potent inducers of type 1
immunity (14). We purified T cells from naive BALB/c mice or from
BALB/c recipients of B6 skin allografts and cultured them in the
presence of B6 splenic stimulators with exogenous IL-4 and
anti-IFN- Ab (XMG1.2). The resultant primed T cell populations
were then studied in recall ELISA spot assays on day 5 (Fig. 3). Pilot studies revealed a maximal
effect of the exogenous IL-4 at a concentration of 500 U/ml and further
revealed that the addition of blocking anti-IL-12 Abs (15.1 and
15.6, kind gifts of Georgio Trinchieri, Wistar Institute, Philadelphia,
PA) had no additional effects beyond that of IL-4 and anti-IFN-
alone (data not shown). We initially postulated that the T cells
obtained from naive mice would be readily polarizable toward type 2
cytokine production as the cells expressed features of undifferentiated
naive T cells (Fig. 1) (33). In contrast, we postulated that
allograft-primed T cells would be more difficult to deviate toward type
2 cytokine production, as many of the responding T cells were already
differentiated into an IFN--producing phenotype.
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. T cells obtained from naive mice
and cultured in the presence of IL-4/anti-IFN- surprisingly
produced a mixture of IFN- (700 spots per 106 cells
plated), IL-4 (5800 spots per 106 cells plated), and
IL-5 (500 spots per 106 cells plated) in a recall assay,
and were not pure type 2 cytokine producers. Furthermore, when T cells
derived from allograft-primed mice were exposed to the same type
2-inducing stimulus, they also produced a high frequency, but mixed
cytokine profile (Fig. 3B). Thus, exogenous IL-4 tended to
shift the overall cytokine profile toward type 2 immunity in both naive
and allograft-primed mice when compared with the naturally developing
cytokine profile (Fig. 2), but additionally primed IFN--producing T
cells in both situations. Type 2 immune deviation readily polarized alloreactive CD4+ but not CD8+ T cells from both naive and allograft-primed mice
We next tested how this immune deviation protocol affected
purified CD4+ and CD8+ T cells from the naive
(Fig. 4) and allograft-primed mice (Fig. 5). Interestingly, for T cells from naive
mice, exogenous IL-4 plus anti-IFN- Ab induced a population of
essentially pure type 2 CD4+ alloreactive T cells
characterized by IL-4 (22,000 spots per 106 cells
plated) and IL-5 producers (6000 spots per 106 cells
plated), and essentially no detectable IFN- or IL-2 producers (Fig. 4A). Third-party cross-reactivity was less than 5% of the
allospecific response (Fig. 4A). In contrast, the same
immune deviation protocol induced a mixed population of allospecific
CD8+ T cells characterized by a high frequency of IFN-
producers (5500 spots per 106 cells plated), in addition
to both IL-4 (3000 spots per 106 cells plated) and IL-5
producers (1500 spots per 106 cells plated) (Fig. 4B). Thus, under identical conditions, this immune deviation
protocol had markedly different effects on the two T cell subsets.
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, A and B). Primed
CD4+ T cells, which produced a mixed cytokine profile when
tested in a primary recall response (Fig. 2), could still be polarized
toward type 2 cytokine production with a low frequency of residual
IFN- producers when recultured in the presence of allostimulators
and IL-4/anti-IFN- Ab. Thus, the allograft-primed
CD4+ T cells were not irreversibly differentiated into type
1 cytokine-producing T cells. The CD8+ T cells from
allograft-primed mice (high-frequency IFN- producers in a primary
recall response; Fig. 2) were further primed to produce a mixed
cytokine profile characterized by IFN-, IL-4, and IL-5 producers
when cultured with allostimulators and IL-4/anti-IFN- Ab (Fig. 5B). Regardless of whether the alloreactive T cells derived
from naive or specifically allograft-primed mice, the CD4+
T cells could be induced to overwhelmingly produce IL-4 and IL-5 with
little to no IFN-, while the same treatment primed CD8+
T cells to produce IFN- in addition to IL-4 and IL-5. Allospecific type 2 cytokine-secreting CD4+ and CD8+ T cells exhibit different functional characteristics
To further define the functional characteristics of our
allospecific, type 2 cytokine-secreting T cells, we evaluated their
ability to mediate DTH responses (Fig. 6,
A and B). The type 2 cytokine-producing
CD4+ T cells (IL-4/IL-5 secreting) induced
alloantigen-specific ear swelling. The injection site was distinguished
by a focal area containing increased numbers of inflammatory cells in
the dermis. Histologic examination confirmed classic findings of
perivascular and dermal edema, and mononuclear cell infiltration with
occasional eosinophils, consistent with DTH (Fig. 6B). The
type 2 immune-deviated CD8+ T cells (IL-4, IL-5, and
IFN- secreting) also induced a modest (although Ag-specific)
ear-swelling response (Fig. 6A). Interestingly, however, the
histology of the reaction differed significantly from the
CD4+ T cell-induced DTH and revealed necrosis at the site
of injection, with polymorphonuclear leukocytes and nuclear dust
consistent with ongoing in vivo cytotoxicity (Fig. 6B).
These findings suggested that the type 2 cytokine-secreting
CD8+ T cells might have distinct functional characteristics
compared with the type 2 cytokine-secreting CD4+ T cells.
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C, the
CD8+ T cells (which produced IFN-, IL-4, and IL-5)
mediated allospecific killing of labeled target cells. In contrast, the
immune-deviated CD4+ T cells were unable to kill any
target, even in an Ag-independent manner (i.e., in the presence of Con
A bridging). In sum, type 2 immune deviation resulted in different
functional outcomes for alloreactive CD4+ versus
CD8+ T cells: the CD4+ cells were strong
mediators of DTH, but not cytotoxicity, while the CD8+
cells were capable of cytotoxicity, but not histologically classic DTH. Both allospecific type 2 cytokine-secreting CD4+ and CD8+ T cells mediate allogeneic skin graft rejection
To test whether the type 2 cytokine-secreting T cells could
mediate allogeneic skin graft rejection, the cells were adoptively
transferred into BALB/c SCID recipients of B6 skin grafts (Table I). All grafts were maintained
indefinitely (>120 days, n = 5) when no adoptive
transfers were performed. Adoptive transfer of either splenic
CD4+ or CD8+ T cells from naive mice
reproducibly led to skin graft rejection by day 12–13. Interestingly,
when the CD4+ T cells were primed in vitro to produce type
2 cytokines, adoptive transfer resulted in graft rejection in all
animals tested, but with delayed kinetics compared with those given
unprimed CD4+ T cells (Table I). Adoptive transfer of
CD8+ T cells primed in vitro to produce type 2 cytokines
also rejected their grafts (Table I), with the type 2
cytokine-producing cells exhibiting delayed kinetics vs the CD8 cells
from naive mice. In additional control experiments, adoptive transfer
of unfractionated T cells from naive mice and unfractionated T cells
primed in vitro to produce type 2 cytokines (Fig. 3) resulted in graft
rejection by day 15 (Table I).
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) confirmed that adoptive transfer of
both the CD4+ and the CD8+ T cells resulted in
graft necrosis, with a complex mixed cellular infiltrate consisting of
lymphocytes, tissue histiocytes, numerous degranulated mast cells, and
focal areas of polymorphonuclear leukocytes associated with necrosis
(Fig. 7, A–D). As IL-4 and IL-5 are known to induce influx
of eosinophils (22, 48), we next sought to specifically determine
whether eosinophils were present in the rejecting skin. Skin graft
samples were stained with a polyclonal antiserum specific for
eosinophil-derived MBP (21, 41). The skin grafts from all five animals
adoptively transferred with type 2 cytokine-secreting CD4+
T cells exhibited striking eosinophilic infiltrates, particularly in
regions adjacent to hair follicles (Fig. 7, E–F). The
rejected grafts obtained from animals transferred with type 2
cytokine-secreting CD8+ T cells also showed occasional
eosinophils (Fig. 7, G–H), but the staining was much less
prominent than in the grafts obtained from the animals injected with
CD4+ T cells. Grafts from animals adoptively transferred
with unfractionated T cells revealed typical findings of skin graft
necrosis without the eosinophil infiltration (not shown).
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A, splenocytes of animals
transferred with type 2 cytokine-secreting CD4+ T cells
contained only CD4+ T cells that produced allospecific IL-4
and IL-5 in the absence of IFN-, confirming the stability of the
induced cytokine profile in vivo. Similarly, recall responses performed
on spleen cells from animals adoptively transferred with type 2
immune-deviated CD8+ T cells contained only
CD8+ T cells that produced the same mixture of IFN-,
IL-4, and IL-5, as noted in the original recall response (Fig. 8B). Recall responses in control animals adoptively
transferred with naive CD4+ and CD8+ T cells
revealed a mixture of IFN-, IL-2, and IL-4 similar to the in vivo
allograft-primed response noted in Fig. 2 (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is
in fact necessary to achieve at least one form of allograft tolerance
(49). The studies described in this work provide evidence that allospecific IL-4- and IL-5-producing T cells responding to alloantigens via the direct recognition pathway do not induce allograft tolerance, but instead mediate graft rejection. Although these experiments do not address the role of type 2 cytokine-secreting cells responding to the indirect pathway, we noted similar results upon induction of type 2 immunity toward a single immunodominant, self-restricted allopeptide in another model system (indirect recognition) (37). Our findings also provide some insight into the mechanisms of rejection after type 2 immune deviation, and show that type 2 immune deviation leads to distinctly different effects on alloreactive CD4+ and CD8+ T cells.
In particular, IL-4 plus anti-IFN- Ab induced essentially
unipolar, allospecific type 2 cytokine-secreting CD4+ T
cells (Fig. 3) that were capable of mediating DTH, but not cytotoxicity
(Fig. 6). In contrast, allospecific CD8+ T cells primed
in the presence of anti-IFN- Ab and IL-4 produced IFN- as
well as IL-4 and IL-5 (Fig. 3) and were capable of mediating
cytotoxicity, but not typical DTH (Fig. 6). The finding that IL-4 can
prime CD8+ T cells to both produce IFN- and induce CTLs
is not unprecedented, and is consistent with previously reported
effects of IL-4 in other experimental systems (23, 24, 25, 26). In fact, IL-4
was initially described as an inducer of CTL activity (26).
Our studies further demonstrated that both the allospecific IL-4- and
IL-5-secreting CD4+ and CD8+ T cells were
capable of rejecting allografts. The in vitro functional data (Fig. 6)
suggested, however, that the two T cell subsets may have reached the
same endpoint through different effector mechanisms. T cell-mediated
cytotoxicity was most likely the dominant mechanism of the type 2
cytokine-secreting CD8+ cells. In contrast, the lack of
cytotoxicity and the striking eosinophilic infiltrate found in the
IL-4/IL-5-secreting, CD4--mediated graft rejection (Fig. 7) raise the possibility that infiltrating eosinophils themselves
participated in the graft destruction.
Eosinophils are a hallmark feature of helminth infection and
exposure to environmental allergens (48) and have been shown to be
capable of cytolysis in a number of experimental models (21, 41, 48).
The cytotoxic properties of eosinophils are derived primarily from
their granules, which contain several preformed cytotoxic proteins,
including eosinophil MBP, eosinophil cationic protein,
eosinophil-derived neurotoxin, and eosinophil peroxidase (22, 48).
Eosinophil MBP, found extensively in the grafts rejected by type 2
cytokine-secreting CD4+ T cells (Fig. 7), is a highly
cationic protein that forms the crystalline core of the eosinophil
granule (22, 48). Its likely mode of cytotoxicity is through a
nonspecific interaction with the anionically charged lipid membrane,
causing disruption of the cell membrane and subsequent lysis (21). In
addition to direct cellular cytotoxicity, MBP induces platelet and mast
cell degranulation, thereby exacerbating the inflammatory response
(49). Interestingly, degranulated mast cells were a prominent feature
of the graft destruction induced by both the type 2 cytokine-secreting
CD4+ and CD8+ T cells (Fig. 7).
The data reported herein are consistent with several studies evaluating
the role of type 2 cytokines as mediators of graft rejection. Bishop
and colleagues, for example, showed that type 2 immune deviation,
through inhibition of IL-12 in vivo, led to accelerated graft rejection
in a murine heterotopic cardiac rejection model in which
CD8+ T cells were the dominant effectors (30).
Interestingly, the anti-IL-12 intervention led to an increase in
IL-4, IL-5, and IFN- message in the graft, but did not affect the
number of CTL precursors (30). These findings could be readily
explained by our data: type 2 immune deviation primes CD8+
T cells to become IFN-, IL-4, and IL-5 producers (Fig. 3), and
primes CTL effector function (Fig. 6). Further studies by this group
revealed that CD8-depleted animals exhibited marked eosinophilic
infiltration of cardiac allograft tissue (30, 31), suggesting that the
remaining CD4+ alloreactive T cells functioned similarly to
those induced in our studies. In experiments performed by VanBuskirk et
al., adoptive transfer of alloantigen-specific, CD4+ T
cells that produced IL-4, but not IFN-, led to accelerated
heterotopic cardiac rejection (32), further establishing that type 2
cytokines can mediate the rejection process under certain conditions.
How can these findings, that IL-4- and IL-5-producing alloreactive T cells mediate rejection, be reconciled with the many studies showing an association between type 2 cytokines and the presence of allograft tolerance (15, 16, 17, 18, 19)? Importantly, the majority of these previous studies have demonstrated that type 2 cytokines, in particular IL-4, were detectable in tolerant animals, but the source of the detected cytokines and their role as mediators of tolerance were as not clearly defined. As many cell types other than T lymphocytes can produce these cytokines (14), detection of RNA message may reflect APC secretion and not an Ag-specific type 2 alloimmune response. It remains possible that the IL-4 detected in these other experimental models was derived from non-T cells in a bystander response and that the alloreactive T cells in the tolerized animals were not expanding or producing cytokines at all. Alternatively, our studies showed a prominence of IL-5 production within the population of allospecific, type 2-deviated T cells. The presence or absence of IL-5 in the previously published tolerogenic protocols (15, 16, 17, 18, 19) has not been well established, however. It therefore remains possible that the IL-5, a potent mediator of eosinophil influx and activation (14, 41), may play a critical role in triggering rejection, while preferential production of IL-4 (and/or IL-10) in the absence of IL-5 may lead to tolerance. Analogous studies of allograft rejection using type 2 immune-deviated CD4+ T cells produced from IL-5 knockout mice may be required to fully address these issues.
Our studies also reveal that alloreactivity in naive mice initially
derives from a relatively low frequency of IL-2-producing
CD4+ T cells. This finding, in conjunction with previous
work by our laboratory showing that the alloresponse derives from the
L-selectinhigh population, confirms that alloreactivity
initiates from T cells with a naive, and not an environmentally
preprimed, phenotype (33). Both the CD4+, and the
CD8+, T cells were capable of producing a mixture of type 1
and type 2 cytokines when primed in vitro (Fig. 2) or in vivo (Fig. 3),
but only the CD4+ T cell population responded initially.
These findings are consistent with the hypothesis that in fully
allogeneic graft rejection, the T cell response initiates in the
CD4+ subset, but then rapidly differentiates and spreads to
include a mixed population of CD4+ and CD8+ T
cells (36). The data are further consistent with the suggestion that
CD4+ T cells are needed to provide help for differentiation
of alloreactive CD8+ T cells (36).
These experiments also demonstrate that alloreactive IFN- production
was derived from CD8+ T cells, and not from
CD4+ T cells, during allograft rejection. This
unanticipated result was noted in two fully MHC-disparate mouse strains
(BALB/c, Fig. 2, and C57BL/6, not shown). Similar findings were noted
during rejection of cardiac allografts (30).
Although the primary function of CD8+ T cells has largely
been assumed to be direct cytotoxicity of the transplanted tissue, the
production of IFN- by these cells suggests they may have other
functional characteristics in vivo as well. The IFN- could
contribute to up-regulation of MHC II and costimulatory molecule
expression on the transplant, thus reducing activation thresholds for
infiltrating T cells, with resultant spreading of the alloreactive T
cell repertoire. In addition, the secreted IFN- may activate
macrophages, and thus provide alternative effector mechanisms of graft
destruction, including induction of CD4+ T cell-mediated
DTH and macrophage release of TNF.
In conclusion, our studies definitively show that treatment with IL-4
and anti-IFN- Abs has different effects on alloreactive
CD4+ and CD8+ T cells, and further demonstrates
that allospecific IL-4/IL-5-producing T cells are not tolerogenic.
Instead, both CD4+ and CD8+ T cells secreting
IL-4 and IL-5 reject allografts and may do so by employing different
effector mechanisms. This documented plasticity of the alloimmune
response implies that approaches other than cytokine immune deviation
will be required to achieve the evasive goal of inducing allograft
tolerance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter S. Heeger, Cleveland Veteran’s Affairs Medical Center 111K(W), 10701 East Blvd., Cleveland, OH 44106. E-mail address:
3 Abbreviations used in this paper: MBP, major basic protein; DTH, delayed-type hypersensitivity; PE, phycoerythrin.
Received for publication March 26, 1998. Accepted for publication July 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155:2928.[Abstract]
. Transplantation 62:1908.[Medline]
-producing cells. J. Immunol. 158:643.[Abstract]
is critical for long term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol. 160:2059.This article has been cited by other articles:
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|
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 |
|
 R P Anderson, D A van Heel, J A Tye-Din, M Barnardo, M Salio, D P Jewell, and A V S Hill T cells in peripheral blood after gluten challenge in coeliac disease Gut, September 1, 2005; 54(9): 1217 - 1223. [Abstract] [Full Text] [PDF]
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|
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|
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|
|
|
|
A. Yamada, A. D. Salama, M. Sho, N. Najafian, T. Ito, J. P. Forman, R. Kewalramani, S. Sandner, H. Harada, M. R. Clarkson, et al. CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo J. Immunol., February 1, 2005; 174(3): 1357 - 1364. [Abstract] [Full Text] [PDF]
|
|
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|
 |
|
 S. L. Hargrave, C. Hay, J. Mellon, E. Mayhew, and J. Y. Niederkorn Fate of MHC-Matched Corneal Allografts in Th1-Deficient Hosts Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1188 - 1193. [Abstract] [Full Text] [PDF]
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|
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|
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|
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|
|
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|
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|
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P. S. Heeger, N. S. Greenspan, S. Kuhlenschmidt, C. Dejelo, D. E. Hricik, J. A. Schulak, and M. Tary-Lehmann Pretransplant Frequency of Donor-Specific, IFN-{gamma}-Producing Lymphocytes Is a Manifestation of Immunologic Memory and Correlates with the Risk of Posttransplant Rejection Episodes J. Immunol., August 15, 1999; 163(4): 2267 - 2275. [Abstract] [Full Text] [PDF]
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