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Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While the role of donor-specific Abs in effecting corneal graft rejection is in considerable dispute (11, 12), there is general agreement that T lymphocytes are the most important mediators of cornea graft rejection. In this regard, corneal allografts resemble other types of solid tissue grafts. However, in orthotopic corneal grafting minor histocompatibility (H)3 Ags, rather than Ags encoded within the MHC, appear to be the most important initiators of alloimmunity (1, 13). Whether placed in low risk or high risk eyes, minor H-incompatible cornea grafts are rejected more often and vigorously than are corneas displaying only MHC-encoded alloantigens. This unusual circumstance is due, on the one hand, to the low expression of MHC-encoded molecules on cells of the cornea (4, 14, 15) and, on the other hand, to the absence from the normal cornea of bone marrow-derived dendritic cells and macrophages that function in other solid tissue grafts as "passenger leukocytes" (4, 16). An important consequence of this situation is that recipients of corneal allografts develop delayed hypersensitivity T cells (TDH) directed at minor, rather than major, histocompatibility Ags (17). Moreover, although there is a strong correlation between the emergence of donor-specific cytotoxic T cells (Tc) and graft rejection, direct and indirect experimental evidence implicates TDH rather than Tc as the primary mediators of orthotopic corneal graft rejection (18).
If rejection of orthotopic corneal allografts is due chiefly to the
actions of TDH, then strategies designed to inhibit cells
of this type should have a salutary effect on corneal allograft
survival. Delayed hypersensitivity is typically mediated by a subset of
CD4+ T cells that secrete IFN-
, termed Th1 (19). Cells
of this type have been directly implicated in acute rejection of other
types of solid organ transplants (20, 21). Moreover, Th1 cells are
cross-regulated by a different subset of CD4+ T cells that
secrete IL-4 and IL-10, and are termed Th2. Thus, the cytokines
produced by Th2 cells suppress the activation and release of cytokines
from Th1 cells (19), thereby limiting the ability of the latter cells
to mediate effector responses such as delayed hypersensitivity. Several
forms of transplantation tolerance (22, 23), including neonatally
induced tolerance (24, 25) and the tolerance induced by treatment with
anti-CD4 Abs, have been strongly associated with Th2 cells (26),
implying that regulation of Th1 cells by Th2 cells can promote graft
acceptance.
The systemic immune responses of adult mice can be biased in the direction of Th1 or Th2 cells depending upon the method of initial immunization (27, 28, 29, 30). Moreover, mice that have mounted Th2-type responses to one Ag often display Th2 responses to subsequent immunizations with different Ags (31). We have employed such a strategy in an effort to modify the rate of rejection of orthotopic corneal allografts in mice. Our results indicate that mice with immune systems heavily biased toward Th2 responses accept orthotopic corneal allografts at a higher rate than normal mice. Moreover, orthotopic cornea allograft-bearing, Th2-biased mice acquire regulatory T cells that secrete Th2-type cytokines and promote graft acceptance when adoptively transferred into naive recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from Taconic Farms (Germantown, NY), and adult male BALB.B (C.B10-H2b/LilMcdJ, H-2b), DBA/2 (H-2d), and B10.D2 (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as experimental subjects or cornea graft donors between 8 and 12 wk of age. All animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Antigens
OVA (Sigma, St. Louis, MO) and keyhole limpet hemocyanin (KLH) of Megathura crenulata (Calbiochem, San Diego, CA) were used.
Preimmunization with KLH
To prepare Th2-biased recipients, BALB/c and C57BL/6 mice received i.p. injections of 50 µl of KLH emulsified in IFA (Difco, Detroit, MI). Control mice received HBSS plus IFA.
Secondary immunization
In one set of experiments, BALB/c mice and C57BL/6 mice received into the nape of the neck or into the hind footpad injections of 100 µg of OVA (with 50 µg of KLH or HBSS alone) in CFA. In another set of experiments, allogeneic corneas (full thickness, 2 mm in diameter) were inserted under the skin of the dorsum of one hind foot. Immediately thereafter, the footpad of the same foot received an injection of KLH (50 µg) or HBSS alone in CFA. In a third set of experiments, mice that received an orthotopic corneal allograft into one eye also received into the nape of the neck or into one hind footpad immediately thereafter an injection of KLH (50 µg) or HBSS alone in CFA.
Induction and grading of corneal neovascularization
Corneal neovascularization (referred to as high risk graft beds) was induced by intrastromal sutures as described previously (6). Briefly, under aseptic microsurgical technique using an operating microscope three interrupted 11-0 sutures were placed in the central cornea of one eye of a normal BALB/c mice. Two weeks later, the neovascularized beds then received orthotopic corneal transplants as described below. The neovascularized recipient cornea, including the sutures, was removed at this time.
Orthotopic corneal transplantation
As described previously (32), each recipient was deeply anesthetized with an i.p. injection of 3 mg of ketamine and 0.0075 mg of xylazine before all surgical procedures. The central 2 mm of the donor cornea was excised and secured in recipient graft beds with eight interrupted 11-0 nylon sutures (Sharppoint, Vanguard, Houston, TX). Antibiotic ointment was applied to the corneal surface, and the lids were closed for 72 h with an 8-0 nylon tarsorrhaphy. All grafted eyes were examined after 72 h; no grafts were excluded from analysis because of technical difficulties. Transplant sutures were removed in all cases on day 7.
Assessment of graft survival
Grafts were evaluated by slitlamp biomicroscopy twice a week. At each time point grafts were scored for opacification. A previously described scoring system (1) was used to measure the degree of opacification between 0–5+: 0 = clear and compact graft; 1+ = minimal superficial opacity; 2+ = mild deep (stromal) opacity with pupil margin and iris vessels (iris structure) visible; 3+ = moderate stromal opacity with only pupil margin visible; 4+ = intense stromal opacity with the anterior chamber visible; and 5+ = maximal corneal opacity with total obscuration of the anterior chamber. Grafts with an opacity score of 2+ or greater after 3 wk were considered as rejected (immunologic failure); grafts with an opacity score of 3+ or greater at 2 wk that never cleared by 8 wk were also regarded as rejected (1).
Culture medium
Serum-free medium was used for cultures and was composed of RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin (all from BioWhitaker, Walkersville, MD), and 1 x 10-5 M 2-ME (Sigma) and supplemented with 0.1% BSA (Sigma), insulin, transferrin, and selenium, and culture supplement (1 µg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 µg/ml Fe(NO3)3; Collaborative Biomedical Products, Bedford, MA) (33).
Cell culture
Lymph nodes draining immunization or grafting sites were removed
and pressed through nylon mesh to produce a single-cell suspension. T
cells were subsequently purified to >95% by a T cell enrichment
column (R&D Systems, Minneapolis, MN). The T cells were washed,
counted, and resuspended at 4 x 105 in 96-well
plates. In some experiments T cells were stimulated with x-irradiated
(2000 rad) syngeneic splenic adherent cells pulsed with 50 µg/ml of
OVA or KLH. In other experiments T cells were restimulated with
irradiated (2000 rad) allogeneic splenic adherent cells. Cells were
cultured in serum-free medium at 37°C in an atmosphere of 5%
CO2. For the results presented in Figs. 1
and 2,
lymph node cells from two or three animals were pooled and analyzed per
experiment; each experiment was repeated at least three times with
similar results.
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Cultures were pulsed with 0.5 µCi of [3H]thymidine 8 h before termination, and cells were harvested onto glass filters using an automated well harvester (Tomtec, Orange, CT). Radioactivity was assessed by liquid scintillation spectrometry, and the amount was expressed as counts per minute.
IFN-, IL-2, IL-4, and IL-10 assays
Cultures similar to those described above were established and
sustained for 24, 48, or 72 h. At each time point, supernatants
were collected and analyzed for contents of IFN-, IL-2, IL-4, and
IL-10 using ELISA kits according to the manufacturer’s instructions
(Endogen, Cambridge, MA).
Intracellular staining of IFN- and IL-4
T cells that were cultured with stimulator cells for 24 h
were harvested and treated with HBSS supplemented with 5 µg/ml
brefeldin A (Sigma), a compound known to disrupt the Golgi apparatus,
thus inhibiting protein secretion for approximately 6 h.
Phenotypic analysis was performed by staining freshly recovered cells
with Cy-conjugated CD3+ (1 µg mAb/106 cells
in 1 ml of PBS-1% BSA; PharMingen, San Diego, CA) for 40 min at room
temperature in a dark. The cells were then washed twice in PBS-1% BSA.
The pellet was resuspended in 0.5 ml of PermeaFix buffer (Ortho
Diagnostics, Raritan, NJ; diluted to 1/1.75 with distilled water and
supplemented with 0.1% EDTA) and incubated at room temperature for a
further 40 min. The cells were washed twice in PBS-1% BSA and stained
for intracellular cytokines with FITC-conjugated mAb against IL-4-FITC
or IFN--FITC (1 µg/106 cells; PharMingen) for an
additional 40 min at room temperature in the dark. The cells were then
washed twice in PBS-1% BSA and analyzed by flow cytometry (EPICS XL
analyzer, Coulter, Hialeah, FL). For the results presented in Figs. 1
C and 2C, the mean fluorescence intensity (MFI)
± SEM were determined for CD3+, IFN-+, or
IL-4+ cells. MFI was based on 5000 measured events. The
significance of each data point was assessed by calculating the
half-peak correlation variance.
Statistical methods
Statistical analyses were performed using Fisher’s exact probability test for proportional rates of rejected allografts. All values of p < 0.05 were deemed significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult mice immunized with KLH in the presence of IFA have been
reported to develop CD4+ T cells that secrete IL-4, IL-5,
IL-6, and IL-10 when stimulated in vitro with KLH (31), i.e., Th2
cells, whereas similar mice immunized with KLH in CFA acquire
KLH-specific T cells that secrete predominantly IL-2 and IFN-, i.e.,
Th1 cells. Moreover, mice immunized first with KLH plus IFA respond to
subsequent immunizations with a different protein Ag, e.g., myelin
basic protein plus CFA, and generate CD4+, myelin basic
protein-specific T cells that secrete predominately Th2-type cytokines
(34). Our experimental goal was to use the logic of these experiments
to determine whether corneal allografts placed orthotopically in eyes
of mice with systemic immune systems biased toward Th2 would experience
enhanced survival. To begin these studies, panels of C57BL/6 and BALB/c
mice received an i.p. injection of KLH (50 µg) in IFA (50 µl).
Spleen cells were removed from these mice 4 wk later, rendered into
single cell suspensions, and stimulated in vitro with KLH. The
supernatants of these cultures contained significant quantities of IL-4
and IL-10, but little IL-2 or IFN- (data not shown), indicating that
the mice had mounted a Th2-type response to KLH. To determine whether
mice of this type would respond to a second Ag with a Th2 bias, two
panels of mice were immunized initially with KLH in IFA, then 4 wk
later, one panel received an s.c. injection of KLH (50 µg)
plus OVA (100 µg) mixed with CFA (50 µl). Control panels of mice
immunized initially with KLH in IFA received an s.c. injection of OVA
(100 µg) in CFA. Draining lymph nodes were removed from these mice,
and purified T cells were prepared and stimulated in vitro with
x-irradiated (2000 rad) syngeneic splenic APC pulsed with 50 µg/ml of
OVA. At 24, 48, and 72 h, supernatants were removed from these
cultures and assayed by ELISA for IFN- and IL-4.
[3H]thymidine was added to separate cultures of 72-h
duration to assess T cell proliferation. A third set of cultures was
harvested at 24 h, and the responding T cells were analyzed by
flow cytometry for intracellular content of IFN- and IL-4. The
results of representative experiments are presented in Fig. 1, A–C. As revealed in Fig. 1A, T cells from all
mice proliferated in vitro in response to OVA stimulation. However, the
extent of proliferation was greater with T cells obtained from
KLH-immune mice immunized subsequently with OVA and CFA alone with T
cells from mice that received a subsequent immunization with OVA plus
KLH and CFA. ELISAs of culture supernatants (Fig. 1B)
indicate that unstimulated T cells produced IL-4 spontaneously, but
little if any IFN-. More important, OVA-stimulated T cells from
KLH-immune mice immunized subsequently with OVA alone produced
significantly more IFN- and significantly less IL-4 than
OVA-stimulated T cells from mice whose second immunization contained
both OVA and KLH. Finally, nearly twice as many T cells from KLH-immune
mice that were immunized subsequently with OVA plus KLH responded to
OVA stimulation in vitro by acquiring intracellular IL-4 compared with
T cells from mice immunized with OVA alone, whereas many more T cells
from the latter mice contained intracellular IFN- compared with T
cells from mice immunized with OVA plus KLH (Fig. 1C).
Moreover, the MFI of CD3+ cells containing IFN- after
treatment with OVA alone was significantly greater than that of cells
after treatment with OVA plus KLH. The opposite was true for
CD3+ cells containing IL-4. In aggregate, these findings
confirm the results previously reported by Falcone and Bloom (34) and
indicate that mice that first encounter KLH in the presence of IFA
develop an immune system that is biased toward Th2
responses. When subsequently immunized with a different Ag (OVA) in
conjunction with the original Ag (KLH), the preponderant T cells
responding to OVA (as well as T cells responding to KLH; data not down)
produce IL-4 rather than IFN- and resemble Th2 cells.
In KLH-immune mice, immunization with heterotopic corneal allografts induces donor-specific Th2 cells
Our next goal was to determine whether mice with Th2-biased immune
systems would respond to immunization with alloantigens by developing
alloantigen-specific Th2 responses. Because minor H, rather than MHC,
encoded alloantigens represent the stronger stimulus to alloimmunity
induced by orthotopic corneal allografts, the following experiments
were conducted with C57BL/6 mice as recipients of BALB.B cornea grafts
and with BALB/c mice as recipients of B10.D2 cornea grafts. The members
of these two strain combinations share the same H-2 genes,
but differ at multiple minor H loci. Panels of C57BL/6 and BALB/c mice
received i.p. injections of KLH (50 µg) in IFA. Four weeks later, the
right footpad of one panel of mice received an injection of KLH or HBSS
(control) in CFA. Simultaneously, an incision was made on the dorsal
surface of the same foot, and a segment of BALB.B or B10.D2 cornea (2
mm in diameter) was inserted. In this manner, antigenic information
from both the heterotopic corneal graft and the footpad Ag injection
site would be focused in the draining popliteal lymph node. One week
later, draining popliteal lymph nodes were removed, and T cells,
purified from these lymph node cell suspensions, were stimulated in
vitro with x-irradiated spleen cells from donors genetically identical
with the heterotopic corneal grafts. The results of these experiments
are presented in Fig. 2. Cultures of 72-h duration to which
[3H]thymidine had been added 8 h before termination
revealed T cell proliferation, indicating that recipients of
heterotopic corneal allografts had been primed (Fig. 2A). As
before, T cells from mice that received a footpad injection of HBSS
rather than KLH at the time the allogeneic cornea was placed
heterotopically proliferated more vigorously. Supernatants were removed
from 24-, 48-, and 72-h cultures and assayed for IFN- and IL-4
contents. T cells stimulated with allogeneic spleen cells produced
significant amounts of IFN- (Fig. 2B). The amount of
IFN- produced by T cells from mice that received heterotopic cornea
grafts and KLH into their footpads was significantly less than that
produced by similarly grafted mice that received HBSS injections
instead of KLH. Alternatively, T cells cultured in the presence of
allogeneic spleen cells produced more IL-4 than did T cells cultured in
the presence of syngeneic spleen cells. Moreover, T cells from mice
that received footpad cornea grafts simultaneously with KLH tended to
produce more IL-4 when stimulated with allogeneic spleen cells than did
HBSS-injected controls. In a third assay, T cells were cultured for
24 h in the presence of alloantigenic spleen cells, then harvested
and assessed for intracellular IFN- and IL-4 contents by flow
cytometry. As revealed in Fig. 2C, a significantly higher
percentage of CD3+ cells from mice that received footpad
KLH plus allogeneic cornea grafts contained intracytoplasmic IL-4,
whereas these same populations of CD3+ cells contained a
lower percentage of IFN--containing cells. We conclude from these
results that allogeneic corneal tissue grafted heterotopically into
Th2-biased mice induced donor-specific alloimmunity in which the
responding T cells were also biased toward Th2-type cytokine
production.
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Whereas allogeneic corneas grafted orthotopically into normal
mouse eyes enjoy "immune privilege," similar grafts placed in high
risk eyes are promptly rejected (6). Rejection in the latter instance
is associated with acquisition of donor-specific T cells that mediate
delayed hypersensitivity, a presumed Th1 response. We hypothesized that
allogeneic corneas grafted into high risk eyes of mice with Th2-biased
immune systems would experience enhanced survival. To test the validity
of this hypothesis, panels of BALB/c mice received i.p. injections of
KLH (or HBSS, negative controls) plus IFA. Two weeks later three
sutures were placed in the central cornea of one eye to induce
neovascularization. When new vessel formation was robust (after 2 wk),
these eyes received orthotopic grafts of corneas from either C57BL/6 or
B10.D2 donors. Immediately after the graft procedure was completed,
recipient mice received s.c. injections of KLH or HBSS in CFA placed at
the nape of the neck. The fate of these grafts was then assessed and
scored clinically. The results are presented in Fig. 3, A and B. BALB/c
mice that received i.p. HBSS injections before corneal grafting and
BALB/c mice that received KLH plus IFA before grafting and HBSS at the
time of grafting rejected their orthotopic C57BL/6 cornea grafts with
considerable vigor. However, mice that first received KLH i.p. and then
a second KLH exposure at the time of orthotopic corneal allografting
rejected C57BL/6 grafts less swiftly and less often (Fig. 3A). An even more dramatic enhancement of graft survival was
observed in BALB/c mice that received KLH 4 wk before and again at the
same time as they received an orthotopic B10.D2 corneal allograft. Less
than 50% of these grafts were rejected, and rejection, when it
occurred, was considerably delayed compared with that in mice that
received HBSS at the time of penetrating keratoplasty. These results
support the hypothesis that mice with Th2-biased immune systems
are less able to reject orthotopic corneal allografts than their normal
counterparts.
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. As before, a high proportion of
KLH-immune BALB/c mice that received an orthotopic B10.D2 cornea graft
along with an injection of KLH plus CFA into the nape of the neck
accepted their cornea grafts indefinitely. By contrast, KLH-immune mice
that received only HBSS plus CFA at the time of grafting rejected their
B10.D2 grafts promptly. An intermediate result was observed among
KLH-immune mice that received their second KLH injection at a site
distant from the orthotopic cornea graft. Although fewer of these mice
accepted their cornea grafts than did the mice that received KLH
injections into the nape of the neck, the footpad-injected mice
accepted their orthotopic corneal allografts with a higher frequency
than did mice who only received HBSS rather than KLH at the time of
penetrating keratoplasty. Thus, the ability of a Th2-biased immune
system to influence the survival of orthotopic corneal allografts is
improved if the lymphoid organ handling alloantigenic signals from the
eye is also responding to the Th2-dependent Ag.
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The hypothesis that Th2-biased immune systems promote acceptance
of orthotopic cornea allografts implies that donor-specific T cells are
generated that not only secrete Th2-type cytokines, but down-regulate
the emergence of allodestructive, Th1-type T cells. If true, then T
cells from mice with accepted corneal allografts should be able to
impair graft rejection when adoptively transferred into naive,
syngeneic recipients. To test this implication, panels of BALB/c mice
received KLH plus IFA i.p. Four weeks later, B10.D2 or DBA/2 corneas
were grafted into neovascularized eyes of these mice; simultaneously,
the mice received an injection of KLH (or HBSS) plus CFA into the nape
of the neck. Two weeks later, at a time when the grafts appeared
perfectly healthy, the mice were sacrificed, and their cervical lymph
nodes and spleen were removed. Single-cell suspensions were prepared,
pooled, and injected i.v. (one donor equivalent per recipient) into
naive BALB/c mice. Sutures had already been placed in one eye of these
recipient mice 2 wk previously to create a high risk eye. Immediately
after the transfer of donor lymphoid cells, B10.D2 corneas were grafted
into the neovascularized eyes of adoptive transfer recipients, and the
fate of the grafts was assessed clinically. As the results presented in
Fig. 5 reveal, lymphoid cells obtained
from Th2-biased BALB/c mice bearing healthy B10.D2 corneal allografts
promoted the survival of B10.D2 grafts in high risk eyes of normal
BALB/c mice. If the adoptively transferred cells were obtained from
donors that received a booster injection of HBSS rather than KLH at the
time of cornea grafting, no improved graft survival was observed.
Moreover, lymphoid cells obtained from Th2-biased BALB/c mice bearing
healthy DBA/2 corneal allografts (which share the same MHC Ags, but
display third-party minor Ags) did not promote the survival of B10.D2
grafts in high risk eyes of normal BALB/c mice. These results indicate
that mice that received orthotopic corneal allografts at a time when
their immune system was biased toward Th2 responses acquired
donor-specific regulatory T cells that suppressed the emergence of
allodestructive T cells of the Th1 type.
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To test whether KLH-immune mice that accepted orthotopic corneal
allografts possessed donor-specific T cells of the Th2 type, BALB/c
mice were immunized first with KLH plus IFA i.p. Two weeks later
sutures were placed through the central cornea of one eye and after
another 2-wk interval, B10.D2 corneas were grafted into these
neovascularized eyes. Simultaneously, the mice received into the nape
of the neck KLH (or HBSS) plus CFA. After another 2 wk, draining
cervical lymph nodes and spleens were removed from these mice, and
purified T cells were obtained. These cells were placed in culture and
stimulated with x-irradiated B10.D2 spleen cells. As before, the
cultured cells were assayed for proliferation, cytokine secretion into
the culture supernatant, and intracytoplasmic contents of IFN- and
IL-4. The results of these experiments are presented in Fig. 6 and indicate that T cells from
cornea-grafted mice that received immunization with CFA plus HBSS
proliferated vigorously, and secreted copious amounts of IFN- when
stimulated with donor alloantigens in vitro. By contrast, T cells from
cornea-grafted mice immunized with CFA plus KLH proliferated less well
and secreted IL-4, rather than IFN-, when stimulated with donor
alloantigens. Neither T cell type produced sufficient TGF-ß in these
experiments to be detectable in our assays (data not shown). We
conclude that KLH-immune mice with Th2-biased immune systems respond to
alloantigens on orthotopic cornea grafts by generating Th2-type
alloreactive T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
TNF-ß in response to Ag recognition have emerged as key effectors in
rejection of solid tissue allografts (20, 21). The central role in
graft rejection of Th1-type CD4+ T cells that recognize
allogeneic class II MHC molecules derives in part from the prominent
role of passenger leukocytes typically present in solid tissue grafts.
Bone marrow-derived dendritic cells and macrophages that constitutively
express class II molecules confer potent immunogenicity to solid tissue
grafts, in part by activating class II-specific and class II-restricted
CD4+ T cells. The ability of passenger leukocytes to
provide potent costimulation via secretion of IL-12 and expression of
CD40 (35, 36) accounts for why the responding T cells are of the Th1,
delayed hypersensitivity type. Not only is this class of T cells
capable in its own right of triggering an allodestructive inflammatory
response, but Th1 cells secrete cytokines that promote the terminal
differentiation at the graft site of primed, alloreactive cytotoxic
CD8+ T cells. Consequently, the combined actions of
CD4+ and CD8+ effector T cells, both of which
arise from Th1 cells activation, effect acute graft rejection. On the other hand, Th1 and Th2 cells each secrete distinctly different arrays of cytokines with widely different consequences (19, 37, 38, 39). Th1 cells promote immunogenic inflammation, not only via their own direct ability to secrete proinflammatory cytokines, but because they promote B cell differentiation in a manner that generates Igs of the complement-fixing variety. Th2 cells, by contrast, do not usually mediate inflammatory responses directly (although exceptions to this statement exist), and the influence of these T cells on B cell differentiation causes the emergence of cells that secrete IgG Abs that do not fix complement. These properties have given Th2 cells the reputation of being anti-inflammatory. In many ways, this reputation is well deserved because Th2 cells possess the unique capacity to down-regulate the development and expression of Th1-dependent immunity (19). In fact, the unique array of cytokines produced by Th2 cells, including IL-4 and IL-10, directly inhibits Th0 cells from differentiating into Th1 cells and suppresses Th1 cells from producing their distinctive cytokines. Similarly, Th1 cells can inhibit the generation and functions of Th2 cells (40).
Experimental evidence from a variety of laboratories indicates that mice that develop a strong Th1-type response to one Ag respond to subsequent Ags with a Th1-biased response. Conversely, mice that respond to one Ag with a vigorous Th2-type response respond to subsequent Ags with a similar Th2-biased response. In particular, Falcone and Bloom (34) have reported that immunization against a non-self Ag that leads to a Th2-type response biases recipients such that subsequent immunization with an autoantigen fails to result in autoimmune disease. The lack of disease in this case correlates with autoreactive T cells that secrete Th2-type cytokines. Our experimental results confirm these findings and indicate that the immune apparatus can be experimentally biased such that subsequent immune responses to other Ags will be predominately of the Th2 (or of the Th1) type. Not all investigators agree that Th1-type responses promote, whereas Th2-type responses suppress, allograft rejection. Thus, Piccotti et al. (41) have reported that inhibition of IL-12 activity, which promotes Th2 responses, nonetheless enhances the rejection of cardiac allografts. Moreover, VanBuskirk et al. (42) have found that infusion of Th2-biased T cells into SCID mice bearing cardiac allografts precipitates acute graft rejection. Thus, it cannot be simply assumed that biasing an alloimmune response toward the Th2 direction will necessarily promote graft survival. Other factors must govern the outcome, and our experiments suggest that immune privilege of the graft site may be one of those factors.
Results from experiments in numerous laboratories indicate that rejection of orthotopic corneal allografts in rodents is mediated predominately by CD4+ T cells of the delayed hypersensitivity, Th1 type (18, 43). Since Th2 cells can down-regulate Th1 cells (19), pre-emptive biasing of a recipient’s immune system in a Th2 direction should have a salutary effect on corneal allograft acceptance. Our experimental results indicate that this is indeed the case. C57BL/6 and BALB/c mice immunized first with KLH plus IFA developed immune systems biased toward Th2 responses. When immunized subsequently with minor H-incompatible alloantigenic corneal tissue, the T cells of these mice responded in vitro to donor alloantigens by producing predominantly Th2-type cytokines. Our experiments indicate that this pattern of cytokine production was promoted by preliminary biasing of recipient immune systems with KLH and IFA because the effect was only observed if subsequent immunization with alloantigenic tissue was accompanied by re-exposure to KLH. Thus, CD4+ T cells that respond to minor H Ags resemble CD4+ T cells that recognize soluble protein Ags in their penchant to differentiate into IL-4-secreting cells when they encounter their Ag in a microenvironment containing activated Th2 cells. This point was strongly supported by the fate of allogeneic corneas grafted into high risk eyes of mice with KLH-induced Th2-biased immune systems. KLH-primed BALB/c mice accepted a very high proportion of orthotopic B10.D2 cornea grafts, whereas control mice rejected such cornea grafts acutely. It is of interest that the success of grafts in KLH-immune mice was significantly enhanced if the cervical lymph nodes draining the graft site received a booster injection of KLH at the time of grafting. This result emphasizes the importance of a Th2-like microenvironment in the draining lymph node at the time donor-specific T cells are first activated.
Our findings also indicate that even MHC-incompatible corneal allografts displayed enhanced survival when placed in high risk eyes of KLH-immune BALB/c mice, although the effect was less pronounced than when minor H only, disparate grafts were used. The enhanced survival displayed by cornea grafts in eyes of Th2-biased mice was clearly related to the emergence of donor alloantigenic-specific T cells of the Th2 type. Not only did T cells harvested from these mice resemble Th2 cells when stimulated with donor alloantigens in vitro, but they were able to suppress the rejection of orthotopic corneal allografts when adoptively transferred into naive, syngeneic mice. We conclude that pre-emptive biasing of a recipient immune system toward the Th2 phenotype is an effective strategy for promoting the acceptance of corneal allografts, even grafts placed in high risk eyes with neovascularized corneas.
A vast literature is accumulating that links Th2 responses to
successful solid organ transplants (20, 21, 22, 26, 44). An early
indication that IL-4-producing, rather than IL-2-producing,
CD4+ T cells contributed to allograft tolerance was
reported from our laboratory in 1990 (24). We demonstrated in neonatal
transplantation tolerance that the CD4+ T cells that
promote long term acceptance of class II MHC disparate skin allografts
not only secreted IL-4 in response to the tolerated alloantigens, but
these T cells also suppressed IL-2 production by alloreactive T cells
in cocultures. Shortly thereafter, other investigators reported that
the alloreactive T cells of rats bearing long accepted cardiac
allografts secreted Th2-type cytokines. Subsequently, other forms of
transplantation tolerance (22, 23, 44), including oral tolerance (45)
and unresponsiveness induced by monoclonal anti-CD4 and anti CD8
Abs (26, 46), were also found to correlate with the emergence of
donor-specific T cells that secreted Th2 cytokines. Thus, among the
many forms of experimental transplantation tolerance in which the
unresponsive state is maintained actively, donor-specific Th2 cells
seem to emerge and contribute to maintenance of the tolerant state.
However, in our cornea-grafting studies, donor-specific Th2 cells made
a much more substantial contribution to graft success than merely
maintaining an already established state of tolerance. Our findings
indicate that mice with immune systems already biased toward Th2
responses failed to acquire (or only poorly so) donor-specific,
allodestructive CD4+ T cells, i.e., secrete IFN- and
mediate delayed hypersensitivity. Consequently, recipient mice were
largely unable to mount a response capable of destroying their
orthotopic cornea grafts. The capacity to prevent an immunopathogenic
Th1-type response by pre-emptively creating a Th2-biased immune system
offers a potential avenue of immunotherapy with broad applicability. To
this end, Tian et al. have already reported that nasal administration
of glutamate decarboxylase peptides to young NOD mice generates Th2
responses and prevents the emergence of insulin-dependent diabetes
(47).
While the ability of mice with Th2-biased immune systems to accept orthotopic corneal allografts in high risk eyes is impressive, the success of this approach may be inherently greater for this particular type of grafting. Corneal tissue itself expresses the inherent property of immune privilege (2, 3, 5), which means that as a graft it erects barriers to immune rejection. Moreover, orthotopic corneal allografts when placed in the eye form the anterior wall of the anterior chamber, an immune-privileged site. It is widely believed that immune privilege is the reason that orthotopic corneal allografts in humans are by far the most successful of all solid tissue grafts. Such may be the case, and for this reason we used recipient mice whose eyes were neovascularized and thus deprived of immune privilege (48). Both MHC and minor H disparate cornea grafts were rejected acutely in these high risk eyes (13). Our finding that mice with Th2-biased immune systems rejected a higher fraction of MHC plus minor H disparate corneas, compared with minor H only disparate corneas, warrants comment. Cornea tissue differs fundamentally from other solid tissue grafts in being virtually devoid of passenger leukocytes (4, 16). Cornea tissue contains neither Langerhans cells nor other bone marrow-derived cells, and thus does not express class II alloantigens (4, 15, 16, 49). Moreover, expression of class I molecules is very low on corneal endothelial cells and keratocytes (4, 15, 49). For these reasons, minor histocompatibility Ags have been determined to offer stronger barriers to corneal graft acceptance than MHC alloantigens, and the ability of recipient T cells to detect alloantigens on orthotopic cornea grafts rests primarily with the capacity of recipient APC to migrate into the graft (50). Thus, the majority of T cells that effect corneal allograft rejection detect and respond to graft-derived alloantigens via the so-called indirect pathway of allorecognition (51, 52, 53). These responding T cells are mostly CD4+. In our current experiments, we have assumed that the Th2 bias of recipient immune systems influenced donor minor H-specific, self-MHC restricted CD4+ T cells to differentiate into Th2-type cells, thereby preventing the emergence of donor-specific Th1 cells that would have mediated graft rejection. The further finding that minor H only disparate cornea allografts survived better in Th2-biased mice than grafts that also displayed MHC alloantigens supports this view. Not only are the T cells that recognize MHC encoded alloantigens via the direct pathway of allorecognition of higher frequency in naive mice than minor H-specific T cells, but many of the direct alloreactive T cells have the phenotype of memory Th1-type cells and may, therefore, be less susceptible to being deviated in a Th2-biased environment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford St., Boston, MA 02114. E-mail address:
3 Abbreviations used in this paper: H, histocompatibility; TDH, delayed hypersensitivity T cells; Tc, cytotoxic T cells; KLH, keyhole limpet hemocyanin; MFI, mean fluorescence intensity.
Received for publication November 3, 1998. Accepted for publication February 8, 1999.
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, C57BL/6)
or OVA, KLH, and CFA (
, experimental, BALB/c; 
, C57BL/6). Seven
days later, the mice were killed; purified T cells prepared from
draining lymph nodes were stimulated in vitro with OVA for 3 days. To
assess proliferation (A), [3H]thymidine
was added during the terminal 8 h of culture. To assess IFN-
), or with KLH plus IFA followed by KLH
plus CFA at the time of grafting (n = 10; 
