Mice with Th2-Biased Immune Systems
Accept Orthotopic Corneal Allografts Placed
in ''High Risk'' Eyes
This information is current as
of July 31, 2017.
Jun Yamada, Munenori Yoshida, Andrew W. Taylor and J.
Wayne Streilein
J Immunol 1999; 162:5247-5255; ;
http://www.jimmunol.org/content/162/9/5247
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References
Mice with Th2-Biased Immune Systems Accept Orthotopic
Corneal Allografts Placed in “High Risk” Eyes1
Jun Yamada, Munenori Yoshida, Andrew W. Taylor, and J. Wayne Streilein2
A
significant number of allogeneic corneas grafted orthotopically into normal eyes of normal mice and rats are
accepted indefinitely (1). This high rate of acceptance,
compared with the virtually uniform rejection of orthotopic allografts of other solid tissues, is an expression of the existence of
immune privilege in the eye (2–5). When immune privilege in the
recipient eye is compromised (so-called high risk eyes), orthotopically placed allogeneic cornea grafts are promptly rejected (6).
These results from experiments with laboratory animals faithfully
reflect the experience with penetrating kelatoplasties in human beings. Allogeneic corneas placed in low risk human eyes display a
high rate of acceptance, and rejection episodes are usually treated
successfully with only topical immunosuppressive therapy (7, 8).
By contrast, cornea grafts placed in high risk human eyes, e.g.,
eyes with scarred or inflamed corneal surfaces, have a very poor
prognosis, and even intensive systemic immunosuppressive therapy is often of no avail (9, 10). Since penetrating keratoplasties are
the most common type of transplant performed in humans, and
since failure of cornea grafts in high risk eyes is an important cause
of blindness, understanding the pathogenesis of graft rejection as a
preamble to developing successful antirejection therapies is a worthy goal for research.
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
Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114
Received for publication November 3, 1998. Accepted for publication February
8, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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 CD41 T cells
that secrete IFN-g, 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 CD41 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 antiCD4 Abs, have been strongly associated with Th2 cells (26),
implying that regulation of Th1 cells by Th2 cells can promote
graft acceptance.
1
This work was supported in part by National Institutes of Health Grant EY10765
and by a generous gift from Mr. and Mrs. Harry Axelrod.
2
Address correspondence and reprint requests to Dr. J. Wayne Streilein, Schepens
Eye Research Institute, 20 Staniford St., Boston, MA 02114. E-mail address:
[email protected]
Copyright © 1999 by The American Association of Immunologists
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.
0022-1767/99/$02.00
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CD41 T cells of the Th1 type play a central role in acute rejection of solid tissue grafts, including orthotopic corneal allografts.
Th1 cells, which mediate delayed hypersensitivity, are the polar opposites of CD41 Th2 cells, and the latter cells cross-regulate Th1
cells through the unique pattern of cytokines they secrete. As such, Th2 cells may have a useful role to play in preventing rejection
of corneal allografts. To test this possibility, the immune systems of adult mice were biased toward Th2 responses by immunization
with keyhole limpet hemocyanin plus IFA. When immunized subsequently with either OVA or allogeneic corneal tissue, these mice
acquired Ag-specific primed T cells of the Th2 type. More important, allogeneic corneas grafted into neovascularized eyes of
Th2-biased mice experienced significantly enhanced survival. To demonstrate that enhanced survival was promoted by donorspecific Th2 cells, lymphoid cells from keyhole limpet hemocyanin-immune mice bearing healthy corneal allografts suppressed
orthotopic corneal allograft rejection when adoptively transferred into naive, syngeneic recipients. We conclude that acceptance
of corneal allografts in neovascularized mouse eyes can be significantly enhanced by biasing the recipient immune system toward
Th2 responses. The Journal of Immunology, 1999, 162: 5247–5255.
5248
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 (27–30). 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.
Materials and Methods
Animals
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 ml 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 mg of OVA
(with 50 mg 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 mg) 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 mg) 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.
opacity; 21 5 mild deep (stromal) opacity with pupil margin and iris
vessels (iris structure) visible; 31 5 moderate stromal opacity with only
pupil margin visible; 41 5 intense stromal opacity with the anterior chamber visible; and 51 5 maximal corneal opacity with total obscuration of
the anterior chamber. Grafts with an opacity score of 21 or greater after 3
wk were considered as rejected (immunologic failure); grafts with an opacity score of 31 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 mg/ml streptomycin (all from
BioWhitaker, Walkersville, MD), and 1 3 1025 M 2-ME (Sigma) and
supplemented with 0.1% BSA (Sigma), insulin, transferrin, and selenium, and
culture supplement (1 mg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3
ng/ml Na2Se, and 0.2 mg/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 3 105 in 96-well plates. In some experiments T cells were
stimulated with x-irradiated (2000 rad) syngeneic splenic adherent cells
pulsed with 50 mg/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.
Proliferation assay and mixed lymphocyte reactions
Cultures were pulsed with 0.5 mCi 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-g, 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-g, IL-2, IL-4, and IL-10 using ELISA kits
according to the manufacturer’s instructions (Endogen, Cambridge, MA).
Intracellular staining of IFN-g and IL-4
T cells that were cultured with stimulator cells for 24 h were harvested and
treated with HBSS supplemented with 5 mg/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 CD31 (1 mg 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-g-FITC (1 mg/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. 1C and 2C, the mean fluorescence
intensity (MFI) 6 SEM were determined for CD31, IFN-g1, or IL-41
cells. MFI was based on 5000 measured events. The significance of each
data point was assessed by calculating the half-peak correlation variance.
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 –51: 0 5 clear and compact graft; 11 5 minimal superficial
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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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.B10H2b/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.
Th2 CELLS PROMOTE CORNEAL GRAFT SURVIVAL
The Journal of Immunology
5249
Results
In KLH-immune mice, immunization with OVA induces
OVA-specific Th2 cells
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Adult mice immunized with KLH in the presence of IFA have
been reported to develop CD41 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-g,
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 CD41, 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 mg) in IFA (50 ml). 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-g (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 mg) plus OVA (100 mg) mixed with CFA (50 ml). Control
panels of mice immunized initially with KLH in IFA received an
s.c. injection of OVA (100 mg) 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 mg/ml of OVA. At 24, 48, and 72 h,
supernatants were removed from these cultures and assayed by
ELISA for IFN-g 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-g 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 KLHimmune 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-g. More important, OVA-stimulated T cells from KLH-immune mice immunized subsequently
with OVA alone produced significantly more IFN-g 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-g
compared with T cells from mice immunized with OVA plus KLH
(Fig. 1C). Moreover, the MFI of CD31 cells containing IFN-g
after treatment with OVA alone was significantly greater than that
of cells after treatment with OVA plus KLH. The opposite was true
for CD31 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
FIGURE 1. Proliferation responses and cytokine secretion by OVA-stimulated T cells. BALB/c and C57BL/6 mice, immunized first with KLH plus
IFA, were immunized 4 wk later with OVA (F, control, BALB/c; f,
C57BL/6) or OVA, KLH, and CFA (E, experimental, BALB/c; M, 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-g and IL-4 production (B), supernatants were collected at 72 h and
assayed by ELISA. To assess intracellular IFN-g and IL-4 contents (C), CD31
T cells collected after 24-h culture were analyzed by flow cytometry using
appropriate anticytokine Abs. In C, the MFI 6 SEM of CD31 were: IFN-g1
after OVA, 5.96 6 0.1; and after OVA plus KLH, 5.13 6 0.11 (p , 0.005);
the MFI 6 SEM of CD31 were: IL-41 after OVA, 4.96 6 0.14; and after
OVA and KLH, 5.50 6 0.09 (p , 0.005).
5250
Th2 CELLS PROMOTE CORNEAL GRAFT SURVIVAL
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-g and resemble
Th2 cells.
In KLH-immune mice, immunization with heterotopic corneal
allografts induces donor-specific Th2 cells
Orthotopic corneal allografts display enhanced survival in KLHimmune recipients
Whereas allogeneic corneas grafted orthotopically into normal
mouse eyes enjoy “immune privilege,” similar grafts placed in
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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 mg) 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-g and IL-4 contents. T cells stimulated with allogeneic spleen
cells produced significant amounts of IFN-g (Fig. 2B). The amount
of IFN-g 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-g and IL-4 contents by flow cytometry. As revealed in
Fig. 2C, a significantly higher percentage of CD31 cells from mice
that received footpad KLH plus allogeneic cornea grafts contained
intracytoplasmic IL-4, whereas these same populations of CD31
cells contained a lower percentage of IFN-g-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.
FIGURE 2. Proliferation responses and cytokine secretion by alloantigen-stimulated T cells. BALB/c and C57BL/6 mice, immunized first with
KLH plus IFA, were immunized 4 wk later with syngeneic or allogeneic
(B10.D2 or BALB.B, respectively) cornea fragments that were implanted
into the dorsum of the right foot. In the footpad of the same limb, KLH (or
HBSS, control) plus CFA were injected simultaneously. Seven days later
the draining lymph nodes were removed, and purified T cells were cultured
with syngeneic or allogeneic stimulator cells for 3 days. Proliferation (A),
IFN-g and IL-4 secretion in supernatants (B), and intracellular IFN-g and
IL-4 contents (C) were assessed and are displayed as described in Fig. 1.
In C, the MFI 6 SEM of CD31 were: IFN-g1 after B10.D2 grafts, 5.12 6
0.08; and after B10.D2 grafts plus KLH, 4.72 6 0.10 (p , 0.005); the
MFI 6 SEM of CD31 were: IL-41 after B10.D2 grafts, 4.78 6 0.11; and
after B10.D2 grafts plus KLH, 5.18 6 0.07 (p , 0.005).
The Journal of Immunology
5251
FIGURE 3. Fate of orthotopic corneal allografts in KLH-preimmunized
mice. BALB/c mice were immunized with KLH plus IFA or with HBSS
plus IFA (control); 2 wk later sutures were placed through the central
cornea of right eyes to induce neovascularization. Two weeks thereafter
corneas from C57BL/6 or B10.D2 donors were placed orthotopically in
these eyes. Immediately thereafter the mice received an injection of CFA
plus KLH or HBSS into the nape of the neck. Graft rejection was scored
clinically (see Materials and Methods), and the results are presented as
Kaplan-Meier survival curves. A, Recipients of C57BL/6 cornea grafts that
were preimmunized with KLH in IFA followed by HBSS plus CFA at the
time of grafting (n 5 13; M), with HBSS plus IFA followed by KLH plus
CFA at the time of grafting (n 5 8; ‚), or with KLH plus IFA followed by
KLH plus CFA at the time of grafting (n 5 10; E). p, p , 0.009. B,
Recipients of B10.D2 cornea grafts that were preimmunized with KLH
plus IFA followed by HBSS plus CFA at the time of grafting (n 5 13; M)
or with KLH plus IFA followed by KLH plus CFA at the time of grafting
(n 5 14; E). p, p , 0.0001.
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 en-
hancement 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.
In the experiment just described, mice with reduced capacity to
reject cornea allografts received two injections of KLH: the first
with IFA i.p., and the second with CFA injected into the nape of
the neck. This latter site was chosen because the draining cervical
lymph nodes also serve as the draining nodes of eyes bearing orthotopic corneal allografts. To determine whether simultaneous
processing of KLH and alloantigens in the same lymph node is
important to allograft acceptance, similar experiments were performed with the following modification: at the time donor B10.D2
corneas were grafted into high risk eyes, KLH plus CFA was either
injected into the nape of the neck or into one hind footpad. Control
mice received HBSS plus CFA into the nape of the neck. The fate
of these grafts is summarized in Fig. 4. 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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FIGURE 4. Fate of orthotopic corneal allografts in KLH-preimmunized
mice that received a second challenge with KLH at the same or a distant
site. BALB/c mice were immunized with KLH plus IFA or with HBSS plus
IFA (control); 2 wk later sutures were placed through the central cornea of
right eyes to induce neovascularization. Two weeks thereafter corneas from
B10.D2 donors were placed orthotopically in these eyes. Immediately
thereafter the mice received an injection of CFA plus KLH or HBSS either
into the nape of the neck or into one hind footpad. Graft rejection was
scored clinically (see Materials and Methods) and is presented as KaplanMeier survival curves. KLH-presensitized recipients of B10.D2 cornea
grafts were challenged immediately after grafting with HBSS plus CFA
into the nape of the neck (n 5 13; M), with KLH plus CFA into one hind
footpad (n 5 10; ‚), or with KLH plus CFA into the nape of the neck (n 5
18; E). ‚ vs E, p , 0.05; E vs M, p , 0.0001.
5252
Th2 CELLS PROMOTE CORNEAL GRAFT SURVIVAL
Th2-enhanced corneal allograft survival can be adoptively
transferred
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
FIGURE 6. Proliferation responses and cytokine secretion by donorspecific T cells from KLH-preimmunized mice that accepted orthotopic
corneal allografts. KLH-preimmunized BALB/c mice received orthotopic
B10.D2 corneal allografts followed immediately by injection of KLH or
HBSS plus CFA into the nape of the neck. Seven days later the draining
lymph nodes were removed, and purified T cells were cultured with syngeneic or B10.D2 stimulator cells for 3 days. Proliferation (A) and IFN-g
and IL-4 secretion in supernatants (B) were assessed and are displayed as
described in Fig. 1.
toward Th2 responses acquired donor-specific regulatory T cells
that suppressed the emergence of allodestructive T cells of the Th1
type.
Alloreactive T cells from KLH-immune mice that accept
orthotopic cornea grafts display a Th2 phenotype
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-g and IL-4. The results of these experiments are presented in Fig. 6 and indicate that T cells from
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FIGURE 5. Fate of orthotopic corneal allografts in recipients of lymphoid cells from KLH-preimmunized donor mice. BALB/c mice were preimmunized with KLH plus IFA. Four weeks later, these mice received
orthotopic B10.D2 or DBA/2 cornea grafts and simultaneously were immunized in the nape of the neck with KLH or HBSS plus CFA. Two weeks
later, cervical lymph nodes and spleens were removed, and single-cell suspensions were injected i.v. into naive BALB/c mice in which one eye had
suture-induced neovascularization. Immediately after the transfer, B10.D2
corneas were grafted into these neovascularized eyes. Graft rejection was
scored clinically (see Materials and Methods) and is presented as KaplanMeier survival curves. M, Recipients of lymphoid cells from donors challenged with HBSS plus CFA at the time they received B10.D2 cornea
grafts (n 5 10); E, recipients of lymphoid cells from donors challenged
with KLH plus CFA at the time they received B10.D2 cornea grafts (n 5
10); ‚, recipients of lymphoid cells from donors challenged with KLH plus
CFA at the time they received DBA/2 cornea grafts (n 5 10). E vs M, p ,
0.0002; E vs ‚, p , 0.002.
The Journal of Immunology
cornea-grafted mice that received immunization with CFA plus
HBSS proliferated vigorously, and secreted copious amounts of
IFN-g 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-g,
when stimulated with donor alloantigens. Neither T cell type produced sufficient TGF-b 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.
Discussion
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 Th2biased 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 CD41 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, CD41 T cells that respond to minor
H Ags resemble CD41 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 KLHinduced 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 Th2biased 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.
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Attempting to prevent rejection of orthotopic corneal allografts by
biasing the recipient’s immune system toward Th2 responses represents the merging of two independent lines of investigation. On
the one hand, CD41 T cells that mediate delayed hypersensitivity
and secrete the proinflammatory cytokines IFN-g and TNF-b 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 CD41 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 CD41 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 CD81 T cells. Consequently, the combined actions of
CD41 and CD81 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, 37–39). 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 IL10, 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
5253
5254
lorecognition (51–53). These responding T cells are mostly CD41.
In our current experiments, we have assumed that the Th2 bias of
recipient immune systems influenced donor minor H-specific, selfMHC restricted CD41 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 Th1type cells and may, therefore, be less susceptible to being deviated
in a Th2-biased environment.
Acknowledgments
We thank Drs. Bruce Ksander and Reza Dana for helpful discussions of
these experiments and Dr. Andrew W. Taylor for assistance in determining
the statistical significance of the flow cytometry measurements. We acknowledge the excellent managerial support of Dr. Jacqueline Doherty and
Marie Ortega.
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A vast literature is accumulating that links Th2 responses to
successful solid organ transplants (20 –22, 26, 44). An early indication that IL-4-producing, rather than IL-2-producing, CD41 T
cells contributed to allograft tolerance was reported from our laboratory in 1990 (24). We demonstrated in neonatal transplantation
tolerance that the CD41 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 donorspecific 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) donorspecific, allodestructive CD41 T cells, i.e., secrete IFN-g 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
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APC to migrate into the graft (50). Thus, the majority of T cells
that effect corneal allograft rejection detect and respond to graftderived alloantigens via the so-called indirect pathway of al-
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