| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Medicine, University of New South Wales, Liverpool Hospital, Liverpool, New South Wales, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and
TNF-ß, but not for Th2 cytokines IL-4 and IL-6 or the cytolytic
molecules perforin and granzyme A. These hosts accepted PVG skin grafts
but rejected third-party grafts, which were not blocked by
anti-IL-4 mAb. Cells from these tolerant hosts proliferated in MLC
and produced IL-2, IFN-, and IL-4 at levels equivalent to naive
cells. Unfractionated and CD4+ T cells, but not
CD8+ T cells, transferred specific tolerance to irradiated
heart grafted hosts and inhibited reconstitution of rejection by
cotransferred naive cells. This transfer of tolerance was associated
with normal induction of IL-2 and delayed induction of IFN-, but not
with increased IL-4 or IL-10 mRNA. Transfer of tolerance was also not
inhibited by anti-IL-4 mAb. This study demonstrated that tolerance
induced by a nondepleting anti-CD4 mAb is maintained by a
CD4+ suppressor T cell that is not associated with
preferential induction of Th2 cytokines or the need for IL-4; nor is it
associated with an inability to induce Th1 cytokines or
anergy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Th1 cells are activated by IL-12; they produce IL-2, IFN-, and
TNF-ß but not Th2 cytokines; and their activation is inhibited by
IL-4 and IL-10 (28, 29). Th2 cells are activated by IL-4; they produce
IL-4, IL-5, IL-6, IL-10, and IL-13 but not Th2 cytokines; and their
activation is inhibited by IFN- (28, 29). IL-4 has been the most
studied Th2 cytokine, and many of the effects of Th2 cells have been
attributed to this cytokine alone or in combination with IL-10. It is
thought that naive CD4+ T cells (Th0) can mature into
either Th1 or Th2 cells depending upon the environmental stimuli they
are exposed to on activation. In infections such as
Leishmania (30) and in autoimmunity (31, 32), development of
Th2 responses can lead to chronic infection or protect against
autoimmunity because these cells inhibit Th1 responses. These
observations have been adapted to alloimmune responses; however, the
data supporting a prime role for Th2 in all forms of transplant
tolerance are limited.
A role for Th2 cells in the maintenance of tolerance is best described in neonatal tolerance (33, 34) and in post-total lymphoid irradiation tolerance (35, 36), in which a predominance of IL-4-producing cells as well as blocking of tolerance induction by anti-IL-4 mAb therapy (37, 38) have been described. In allograft tolerance induced in adults, Th2 responses have been described as predominate when either depleting anti-CD4 mAb (19, 20) or CD28-B7 blockade (39) is used to induce tolerance. The mechanisms maintaining long term tolerance are less well described. It is a common finding from a variety of models that specific tolerance can be adoptively transferred by CD4+ T cells (15, 40, 41, 42). The finding that the FcR-bearing subset of CD4+ T cells transfers tolerance has been taken as indirect evidence that it is the Th2 subset (19); however, the cytokine production of these cells was not examined. Transfer of anti-CD4-induced tolerance with CD4+ T cells was only partially inhibited by anti-IL-4 mAb (21). Attempts to facilitate induction of tolerance by administering IL-4 and other Th2 cytokines have produced modest effects (43, 44, 45) or none (22, 46, 47). Several recent studies have demonstrated that Th2 cells may effect rejection rather than mediate graft acceptance (48, 49, 50). In this study, we have examined the mechanisms that maintain long term tolerance in rats treated with a nondepleting anti-CD4 mAb, particularly the role of Th2 cells and IL-4.
In the model studied, MRC Ox38 when given for 2 wk posttransplant induces indefinite survival of full MHC-incompatible PVG cardiac allografts in DA rats (5, 47). The induction of tolerance is associated with minor depletion of CD4+ T cells (<20%) and a specific down-regulation of Th2 cytokines IL-4, IL-5, and IL-13 in the first week after transplantation (47). This is associated with a complete inhibition of alloantibody responses, confirming that Th2 responses were inhibited by the anti-CD4 therapy. Th1 responses were not inhibited (47), a finding that differs from several other reports that used depleting anti-CD4 mAb (19, 20).
In this study, we examined the alloimmune responses in rats that had developed tolerance to their grafts. The ongoing immune response within the grafts, their capacity to accept a second donor strain graft when IL-4 was blocked, the in vitro and in vivo reactivity of cells, and the profile of cytokines induced were studied. Infectious tolerance with CD4+ T cells was not accompanied by up-regulation of Th2 cytokines, and transfer of tolerance was not blocked by an anti-IL-4 mAb. There was no evidence that preferential up-regulation of Th2 cytokines, especially IL-4, was required for the maintenance or transfer of tolerance in this model.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DA (RT1a), PVG (RT1c), Lewis (RT1l), F344 (RT1lv1), and Sprague Dawley rats and BALB/c mice were bred and maintained as previously described (47). Operative procedures including heterotopic heart grafts, neonatal heart grafts, irradiation, preparation of single-cells suspensions from lymph node and spleen, and enrichment for CD4+ T cells have all been previously described in detail (11, 51). All experiments were conducted as approved by the animal ethics committee of the University of New South Wales, Sydney, New South Wales, Australia.
Production and administration of mAb
The clone for MRC Ox38 (IgG1), an anti-rat CD4 mAb, and for MRC Ox81 (IgG1), a mAb that blocks rat IL-4 function (52), were a kind gift of Dr. Don Mason (Medical Research Council Cellular Immunology Unit, Oxford, U.K.). The isotype control mAb was A6 (IgG1), which reacts with human but not mouse CD45RO (53). For functional and in vitro studies, mAb was grown in ascites of BALB/c mice primed by i.p. injection of IFA (Sigma-Aldrich, St. Louis, MO) 3 days before injection of clones. Ascites was purified on a DEAE-Sepharose column (Pharmacia, Uppsala, Sweden), and Ab concentration was determined by radial immunodiffusion (54). Animals were treated with four doses of MRC Ox38, MRC Ox81, or A6 at 7 mg/kg i.p. on the day of transplantation and on days 3, 7, and 10. MRC Ox38 therapy given from the day of transplantation leads to indefinite graft acceptance in all DA recipients compared with normal the rejection time of 6–8 days; it coats peripheral CD4+ cells but does not deplete by >20% (5, 47).
MRC Ox81 therapy given as described inhibits IL-4 function in vivo; it
reduces inflammation in experimental uveoretinitis (55), blocks IgG
isotype switch in alloimmune response (45), and alters the immune
response in Heymann nephritis (S.T.S. and B.M.H., unpublished data). To
confirm the presence of active MRC Ox81 mAb in DA rats treated with one
dose of 7 mg/kg, serum was collected at 4 h and 1, 2, 3, and 8
days. This serum was tested for its ability to block rat rIL-4
up-regulation of class II MHC on B cells in vitro, as described (45).
In this assay, rIL-4 was used at 100-fold above the minimum required to
induce class II MHC expression in the assay; 8 µg/ml of MRC Ox81 was
the minimum concentration required to fully block rIL-4 in this assay.
All samples of serum taken from MRC Ox81-treated rats fully blocked
class II MHC induction by rIL-4 (Fig. 1
).
In addition, in DA rats grafted with PVG hearts, the in vivo effect of
MRC Ox81 on the IgG1 isotype of the alloantibody responses was examined
at day 7 using an indirect flow cytometric assay and specific isotype
antiserum, as described (45). The IgG1 isotype was assayed
because it is dependent on IL-4 for induction of class shift (56). In
sera from rats with normal rejection, an IgG1 response was observed
that was enhanced in rats treated with rIL-4, as described (45). This
response was totally inhibited in rats treated with MRC Ox81 (Fig. 2). Controls treated with isotype-matched
mouse mAb (A6) had similar IgG1 alloantibody responses to normal
rejection (data not shown).
|
|
Indirect immunofluorescence staining of single cells and
immunoperoxidase staining of tissue with mAb were performed as
described (57). The anti-rat mAb used included G4.18 (CD3), R7.3
(TCR
ß), W3/25 (CD4), MRC Ox8 (CD8), MRC Ox6 (MHC class II), and
MRC Ox12 (
light chain Ig) and were either purchased from PharMingen
(San Diego, CA) or produced in our laboratory.
Cell preparation and purification
Single-cell preparations from spleen and lymph nodes were resuspended in PBS with calcium and magnesium salts (DAB; Oxoid, Oxford, U.K.) and 10% FCS (Life Technologies, Gaithersburg, MD) as described (5). Cells were separated by an indirect panning technique to deplete unwanted subpopulations with mAb MRC Ox8 or W3/25 as previously described (5). Enriched populations had a purity of 97–99%.
Cell culture
RPMI 1640 (Life Technologies) was supplemented with 100 ng/ml of penicillin and 100 U/ml of streptomycin (Life Technologies), 2 mM glutamine, 5 x 10-5M 2-ME (Sigma-Aldrich), and either 5–10% FCS or 20% Sprague Dawley rat serum. Of the sera from a variety of different rat strains, 20% Sprague Dawley serum was identified as best supporting MLC because it produced low background stimulation and allowed cytokine mRNA analysis with no background in autologous controls.
Mixed lymphocyte culture
Spleen and thymus cells were isolated from rats given 9.5 Gy whole body irradiation 24 h earlier. These cells were used as stimulator cells and were predominantly dendritic cells, containing <1% lymphocytes. This method of preparing stimulators eliminated background levels of mRNA for T cell cytokines for the RT-PCR assays. These stimulators were as effective as in vitro-irradiated spleen cells at one-tenth the number of cells. Lymph node cells from naive rats or allograft recipients treated with MRC Ox38 and sacrificed >100 days posttransplant were cultured at room temperature overnight in 5% FCS-supplemented medium. These cells were then washed and resuspended in 20% Sprague Dawley serum-supplemented medium, as were the stimulator cells. Responder cells were seeded in 96-well U-bottom plates at 2 x 105 cells/ml and stimulators at 2 x 104 cells/ml in a total volume of 200 µl. Quadruplicate wells were cultured for each sample at 37°C in humidified air containing 5% CO2. Proliferation was assessed by pulsing with 0.5 µCi [3H]TdR (Amersham, Arlington Heights, IL) 18 h before harvesting using a Harvester 96 (Tomtec, Orange, CT) and counting in liquid scintillation fluid on a Microbeta Plus scintillation counter (Wallac Oy, Turku, Finland). The stimulation index was calculated as specific response/response to autologous stimulators. Relative response was calculated as specific response - response to autologous stimulators/response to third party minus response to autologous stimulators.
RT-PCR
The methods of mRNA extraction, cDNA synthesis, and PCR have
been described in detail (47, 57). Semiquantitative PCR was performed
either by terminating the reaction every 3–5 cycles or by diluting the
starting cDNA sample 10-, 100-, and 1000-fold. Results were scored as
the cycle or dilution of cDNA at which specific PCR product was first
detected. All samples were standardized by quantitating the RNA by
spectroscopy before RT-PCR and by PCR of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)4 to confirm intact
RNA and consistency of cDNA synthesis. The primers and optimal
conditions for the cytokines IL-2, IL-4, IL-5, IL-10, IFN-, TNF-,
TNF-ß (lymphotoxin), cytolysin (homologue of mouse perforin), and
granzyme A have been described (47, 57). A five-cycle interval detects
10-fold differences, while a three-cycle interval detects 5-fold
differences; this has been previously demonstrated for IL-2, IFN-,
and IL-10 (47), and IL-4 (45). In the assay range tested, increases and
decreases in mRNA for these cytokines can be demonstrated (45, 47, 57).
Similar dilutional studies have confirmed the sensitivity of the assays
for TNF-, TNF-ß, cytolysin, and granzyme A (our unpublished
data).
Statistical analyses
Data were analyzed with ANOVA, and significance was examined by the Bonferroni-Dunn post hoc test (p < 0.005 for significance); for nonparametric unpaired tests, Mann-Whitney U tests were performed (p < 0.05 for significance) using the Statview program for Apple Macintosh.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The grafted hearts of MRC Ox38-treated recipients were examined >100 days posttransplant, all having contracted normally and with no evidence of acute rejection on palpation. Macroscopically, the hearts appeared normal. Histology and immunopathology revealed a diffuse light infiltrate of mononuclear cells as well as scattered small foci of these cells. The infiltrate include CD4+ and CD8+ T cells and macrophages. Vessels in some grafts showed mild intimal hyperplasia and infiltrate of T cells and macrophages consistent with early chronic rejection.
To examine the cytokine profile of this cellular infiltrate, RT-PCR of
mRNA from transplanted heart tissue was undertaken and compared with
that in the recipients own heart and normal heart tissue (Fig. 3). As a positive control, normal
rejecting hearts at day 6 posttransplant were compared. All cDNA
preparations were standardized and when amplified by PCR had the same
level of GAPDH mRNA, detectable at 15 cycles. The long-surviving hearts
had an increase in mRNA for all three Th1 cytokines examined. IL-2,
IFN-, and TNF-ß were not significantly different from normal
rejection levels; levels of IFN- and TNF-ß in rejecting hearts
were significantly above those found in the recipient’s own heart and
normal heart tissue. Each five-cycle difference represents a
10-fold difference in mRNA. In this assay, low levels of nearly all
cytokines are detected in normal tissue, presumably due to the presence
of circulating activated T cells and to the sensitivity of the RT-PCR
technique used. The levels of Th1 cytokines had fallen from that
observed during the induction of tolerance by anti-CD4 therapy (day
6 posttransplant), but were not back to background levels (data not
shown) as previously published (47). In tolerant grafts, mRNA for the
Th2 cytokines IL-4 and IL-6 were not increased above the level observed
in the recipient’s own heart or in normal hearts. The level of IL-4
was not different from that seen in normal rejection but was above that
seen during the induction phase with anti-CD4 mAb, when IL-4 and
IL-5 expression in the transplant was below that in normal or rejecting
hearts (previously published data (47)). In tolerant grafts, IL-6 was
rarely detected and was significantly below the level found in both
rejecting grafts as well as that observed during induction of
tolerance, when this cytokine was increased to levels seen in normal
rejection. IL-10 expression in tolerant grafts was increased above the
level found in the recipient heart and normal heart, but appeared less
than the levels found during acute rejection or the induction phase of
tolerance, although not reaching significance. The mRNA for effector
molecules TNF- and cytolysin in tolerant hearts were at the
background levels found in normal and recipient hearts, and granzyme A
was not detected in tolerant hearts. These findings demonstrated that
within the tolerated graft there is a persistent infiltration of Th1
cells with no evidence of preferential Th2 cell activation or an
increase in cytotoxic cell molecules.
|
DA rats with long surviving PVG allografts after MRC Ox38 therapy
were tested for specific tolerance by application of donor and
third-party Lewis skin grafts. All third-party grafts were rejected in
first set tempo of 10–13 days (data not shown). If PVG skin grafts
were applied 84–100 days postcardiac transplantation, skin graft
survival was prolonged; however, some grafts were rejected in a delayed
fashion (Table I). After 100 days
posttransplant, all PVG grafts skin were accepted and grafts appeared
normal up to 100 days later. To determine whether IL-4 was essential
for the establishment of tolerance to the second grafts, MRC Ox81, a
mAb that blocks IL-4 function in vitro and in vivo, was administered
(see Materials and Methods). This therapy failed to prevent
acceptance of the new graft or affect the function of the
long-surviving cardiac allograft.
|
Lymph node cells from rats with long-surviving grafts (>100 days)
when stimulated in MLC against specific or third-party Lewis responded
by both proliferation and induction of Th1 cytokines. The response from
five separate tolerant hosts was examined, and the kinetics of
proliferation to PVG and Lewis was identical, starting at day 3 and
peaking at days 4 to 5. The proliferative response of cells from
tolerant hosts was half that of naive cells, but the relative responses
and stimulation indices were similar (Fig. 4, A and B) because
naive cells have higher autologous proliferation. These studies
demonstrate there was no loss in the capacity of cells from tolerant
animals to proliferate against specific donor Ag. RT-PCR was performed
to assess cytokine mRNA in MLC 48 h after stimulation, using a
10-fold dilution series of cDNA. These results show that while there
was some variation between animals, there was comparable induction of
IL-2, IFN-, and IL-4 mRNA in tolerant cells against syngeneic,
specific donor, and third-party Lewis stimulators. These levels were
not significantly different from those in control naive cells (Fig. 4C). IL-10 was not increased above high background
expression in autologous controls (data not shown). Taken together,
these in vitro experiments demonstrate that lymphocytes from tolerant
hosts are not deleted, rendered anergic, or diverted to a Th2 response.
|
For these studies, an adoptive transfer assay with whole body
irradiated DA hosts was used. Initial studies transplanted
donor-specific PVG neonatal heart grafts into one ear and third-party
Lewis grafts in the other; thus, each animal had its own internal
third-party control. Irradiation delays rejection to 31 (27, 28, 29, 30, 31, 32, 33, 34, 35, 36) days
(median (range)) for PVG grafts and 26 days (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) for Lewis compared
with normal rejection times of 13 (11, 12, 13, 14, 15) and 12 (10, 11, 12, 13, 14, 15, 16) days,
respectively (Table II). Naive spleen and
lymph node cells partially restore rejection of both grafts to normal.
Cells from rats with long-surviving grafts after MRC Ox38 therapy were
unable to restore rejection of donor strain PVG grafts but did effect
rejection of third-party Lewis grafts, similar to naive cells. A role
for an active inhibitory cell was shown by cotransfer of cells from
tolerant hosts with naive cells. These experiments were done with a
ratio of tolerant cells to naive cells of 4:1, as this ratio has been
shown necessary in other models of tolerance transfer (40, 58). In
these experiments, cells from tolerant animals inhibited the
cotransferred naive cells capacity to effect rejection of PVG grafts,
but third-party Lewis grafts were rapidly rejected.
|
). CD4+ T cells from
tolerant animals failed to restore PVG graft rejection but did effect
third-party F344 rejection. In this model, naive CD4+ T
cells were all that was required to restore graft rejection (11).
Mixing CD4+ T cells from tolerant rats with naive
CD4+ T cells lead to inhibition of the naive cell’s
capacity to effect PVG graft rejection. CD8+ T cells lack
the capacity to restore rejection in irradiated hosts unless sensitized
(11, 59). CD8+ T cells from tolerant hosts failed to effect
either PVG or F344 graft rejection and, when mixed with naive
CD4+, did not inhibit their capacity to effect rejection
(data not shown). Thus, it was concluded that tolerance was associated
with a CD4+ T cell that could transfer tolerance and had an
inhibitory function on naive CD4+ T cells.
|
, IL-4, and IL-10. GAPDH expression was comparable for all
samples. These studies failed to demonstrate any up-regulation of IL-4
in hosts given tolerant cells (Fig. 5).
There was no significant difference in the up-regulation of IL-4 in
heart grafts and lymph nodes of hosts reconstituted with naive cells
compared with tolerant cells. IFN- was significantly less at early
time points in the grafted hearts of hosts given tolerant cells
compared with those given naive cells, but there was no difference in
expression within the draining node. IFN- levels were also reduced
compared with unreconstituted controls at day 9, although by day 12
there was no significant difference between the treatment groups. This
effect was seen only within specific donor (PVG) grafts, with no
difference in IFN- levels between tolerant and naive cell
reconstituted groups for Lewis grafts (data not shown). IL-2 was rarely
detected in heart grafts. IL-10 was readily detected in all samples at
all time points and was not discriminatory between groups (data not
shown). With the exception of IFN- in grafted hearts, the cytokine
expression profiles were similar in both PVG and Lewis allografted
hosts (data not shown). The samples available from neonatal grafts were
small, not permitting analysis of further cytokines, so experiments
with adoptive transfer to hosts with heterotopic adult heart grafts
were also examined.
|
Irradiated hosts with PVG heterotopic grafts were reconstituted
with either 5 x 106 naive CD4+ T cells or
with 2 x 107 tolerant CD4+ T cells and
5 x 106 naive CD4+ T cells. Controls were
not reconstituted with cells. Graft survival was the same as for
the neonatal heart graft model (Table IV). In this model, as few as 5 x
105 CD4+ T cells effect graft rejection
(unpublished data), but 10 times more naive CD4+ T cells
combined with CD4+ T cells from rats with long-surviving
grafts failed to effect specific donor graft rejection. They did effect
third-party rejection (data not shown). As in the neonatal heart graft
model, tolerant cells mixed with naive cells inhibited the
reconstitution of rejection by the naive cells. To examine whether IL-4
was required to inhibit the naive cell response this cytokine was
blocked with MRC Ox81. This did not inhibit transfer of tolerance.
|
, and TNF-ß,
similar to that seen in rats restored with naive cells as well as in
nonreconstituted hosts (Fig. 6). This
suggests that regenerating cells in the host contribute to the
allograft response. Th2 cytokines IL-4 and IL-10 were not increased in
hosts restored with suppressor cells compared with those restored with
naive cells; but these cytokine levels appeared slightly higher
then in the irradiation controls, although this difference did not
reach significance. Surprisingly, there was also induction of the
effector molecules TNF-, cytolysin, and granzyme A. Despite this
identification of an effector Th1 response and cytolytic T cell
molecules, there was no evidence of graft damage in the hosts restored
with suppressor cells or in the irradiation controls.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Differences between the models of transplant tolerance revolve around
the reactivity of cells from tolerant hosts in vitro and the cytokines
that they may produce or be associated with. Three mechanisms have been
examined to explain tolerance; these are clonal deletion, clonal
anergy, or clonal dysregulation of cytokines (in particular, Th2
dominance over Th1). In our model, alloreactive Th1 but not Th2 cells
are activated during induction (47). This study demonstrated that Th1
activation persists in the graft >100 days and was not accompanied by
a late activation of Th2 cells. Further, the response of tolerant cells
in vitro was similar against a specific donor and a third-party
alloantigen, and mRNA for Th1 cytokines IL-2 and IFN- were induced,
indicating that the cells were not anergic. There was no
heightened induction of IL-4 and IL-10 by these cells, indicating that
Th2 cells were not increased. These results are similar to those of
most other studies of transplant tolerance induced in adults in that
cells can proliferate to the donor in MLC (7, 17, 27, 36, 66); only a
few studies show diminished proliferation (42). When cytokine activity
of in vitro-stimulated cells has been assayed, no loss of IL-2 or
enhancement of IL-4 response has been observed (17, 36). Why
essentially normal alloreactivity in vitro does not translate into
rejection effectors in vivo remains an unresolved paradox of this form
of tolerance. Our in vivo studies accord with our in vitro data in that
within the graft, both in the original tolerant host and in the
adoptive hosts rendered tolerant by transfer of cells, there was
evidence of Th1 cell activation, albeit IFN- in the graft of
adoptive hosts, but not in the regional lymph node, was delayed
compared with naive cell reconstituted, unreconstituted, and
third-party grafts. There was no enhanced induction of mRNA for IL-4 or
other Th2 cytokines. This situation is similar to the induction phase
of tolerance in this model, when there is activation and infiltration
of Th1 cells as well as cytolytic cells and activated macrophages, but
there in no graft injury nor induction of cells with mRNA for IL-4 and
IL-5 (47). Why there is no graft injury is not known, but these
findings suggest that the regulation of tolerance may occur at the
final effector phase. The delayed induction of IFN- in the graft but
not in the regional node suggests there may be a reduced infiltration
of Th1 effector cells, which may account for the failure of the
adoptive host to effect rejection. The mechanism by which there is
reduced infiltration of IFN--producing cells was not ascertained by
these studies. Although our studies did not identify anergy as a
mechanism, anergy in CD8+ T cells has been described with
anti-CD4 mAb-induced tolerance (67). This possibility could not be
explored in DA rats because in this strain CD8+ T cells
alone do not proliferate or produce IL-2 without help from
CD4+ T cells (5, 68).
Tolerance induction was demonstrated by the acceptance of donor skin grafts and rejection of third-party grafts. We reasoned that if IL-4 was to play a major role in the regulation of the Th1 cells and in preventing rejection in this model, the rechallenge with skin, which is a more difficult organ to which to induce tolerance, would provide adequate antigenic challenge and overcome the lack of APC in the original graft. In this circumstance, regulator cells producing IL-4 would be most active in preventing rejection of the new graft. However, administration of a large dose of an anti-IL-4-blocking mAb had no effect, and the new skin graft and the original heart grafts continued to function normally for an additional 100 days. This same batch and dose of mAb has blocked IL-4 function in another model of transplant tolerance induction (45) and has altered the response to autoimmune nephritis in rats (S.T.S. and B.M.H., unpublished data). In the current study, it was shown that significant circulating inhibitory levels of MRC Ox81 could be detected up to 8 days after injection of a single dose of MRC Ox81; thus, experimental hosts given 4 doses over a 10-day period would have had sufficient mAb to block any IL-4 function. The capacity of MRC Ox81 to block IL-4 function in vivo was demonstrated by its ability to block IgG1 alloantibody responses even in hosts treated with large doses of rIL-4. In experimental autoimmune uveoretinitis, a similar dose of MRC Ox81 blocked the proinflammatory effect of rIL-4 on this disease and alone lessened the severity of the disease (55). Taken together, these findings suggest that this dose of MRC Ox81 should have blocked IL-4 function in the skin-grafted tolerant host and should have prevented adoptive transfer of tolerance to irradiated hosts if IL-4 played an essential role in the maintenance of tolerance. In the transfer of anti-CD4/anti-CD8-induced tolerance in mice, blocking anti-IL-4 mAb only partially impaired transfer of tolerance in situations in which the ratio of tolerant to naive cells was marginal (21). Anti-IL-4 mAb more readily blocks induction of neonatal transplant tolerance (38, 69), as well as restoring effector responses to Leishmania (30). Combined with the failure to detect increased mRNA for IL-4 and other Th2 cytokines in adoptive hosts given tolerant cells, these data suggested that IL-4, in particular, and Th2 cells were not necessary for the maintenance of tolerance in this model.
This conclusion is at variance with many other studies on transplant tolerance, which report that Th2 cells and IL-4 in particular are central to maintenance of transplant tolerance (19, 20, 39, 42, 70, 71). Many of these studies have observed heightened levels of IL-4 during induction, usually in models in which there was major T cell depletion or during the neonatal period. These observations may be due to the predominance of Th2 responses early during ontogeny. Nondepleting anti-CD4 mAb in MLC also diverts the response to Th2 (72), which suggests that depletion of CD4+ T cells is not the only factor and that species, strain, prior exposure to Ag, and protocol differences may affect whether Th2 responses are induced. However, few others have examined the change in cytokines during the transfer of tolerance. Transfer of posttotal lymphoid irradiation tolerance was associated with activation of both Th1 and Th2 cells, which was similar to that observed in rats restored with naive cells (36). In our studies, the methods of RT-PCR used have the capacity to detect as little as a fivefold increase or decrease in mRNA for IL-4 (45, 47, 57); thus, biologically significant changes in Th1/Th2 cell function could be assayed and would have been detected. Combined with the failure of anti-IL-4 mAb therapy to affect maintenance or transfer of tolerance, these studies suggest that IL-4 is not an essential cytokine for the maintenance of tolerance. IL-4 itself has mixed effects as an agent to induce transplant tolerance, with some reports showing that IL-4 can prolong graft survival (43, 44, 45), while others report no effect (22, 47, 73). Further, Th2 cells have been proposed as effectors of rejection (48, 49), and in IL-4 knockout mice, transplant tolerance can be induced but is prevented by the administration of rIL-4 (74).
The understanding of the mechanisms that maintain tolerance has major potential to promote clinical organ graft acceptance without the need for long term immunosuppression. From the findings of this and other reports, we conclude that the maintenance of tolerance is due to a CD4+ T cell that can suppress the normal alloreactive cell’s capacity to mount an effector response that destroys the allograft. The mechanisms by which these cells mediate their inhibitory effect remain a matter of speculation. One theory is that the cells act as nonproductive alloreactive cells that consume IL-2, thereby starving the potentially active effector cells from this or other cytokines (17). In support of this possibility is the finding that the maintenance of suppressor cell function in vitro is IL-2 dependent (41) and the cells express IL-2R (62). The current study did not examine whether there was a relative deficit of IL-2, but cells from tolerant animals could produce mRNA for IL-2 when stimulated in vitro and did not impede induction of mRNA for IL-2 in adoptive hosts. The second theory involves Th1 cell inhibition by Th2 cells or other immunoregulatory cells. It has recently been reported that there may be other immunoregulatory cells, including Th3 cells, that have different cytokine profiles than Th2 cells and that produce TGF-ß; these are thought to regulate tolerance in experimental allergic encephalitis (65) and in Tr1 cells that inhibit autoimmune colitis and produce IL-10 and IL-5 but not IL-4 (75). Our findings are consistent with the possibility of either of these regulatory cells playing a role in transplant tolerance, and these possibilities are currently under investigation. The data reported here provide evidence that classical Th2 cells and IL-4 are not essential for the maintenance of tolerance. It is possible that IL-4 is a major immunoregulatory cytokine; in this model, however, it was not essential and may have been replaced by other cytokines with similar actions.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Bruce M. Hall, Head-Department of Medicine, Liverpool Hospital, P.O. Box 103, Liverpool, 2170, N.S.W., Australia. E-mail address:
3 Current address: Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 22, New York, NY 10021.
4 Abbreviation used in this paper: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication February 3, 1998. Accepted for publication July 6, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. F. Jessup, H. M. Brereton, P. J. Sykes, M. A. Thiel, D. J. Coster, and K. A. Williams Local Gene Transfer to Modulate Rat Corneal Allograft Rejection Invest. Ophthalmol. Vis. Sci., May 1, 2005; 46(5): 1675 - 1681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. R. Godfrey, D. J. Spoden, Y. G. Ge, S. R. Baker, B. Liu, B. L. Levine, C. H. June, B. R. Blazar, and S. B. Porter Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function Blood, January 15, 2005; 105(2): 750 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. R. Godfrey, Y. G. Ge, D. J. Spoden, B. L. Levine, C. H. June, B. R. Blazar, and S. B. Porter In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures Blood, July 15, 2004; 104(2): 453 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Gray, P. Arosio, and P. Hersey Association of Increased Levels of Heavy-Chain Ferritin with Increased CD4+ CD25+ Regulatory T-Cell Levels in Patients with Melanoma Clin. Cancer Res., July 1, 2003; 9(7): 2551 - 2559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chen, M. S. Ford, K. J. Young, M. I. Cybulsky, and L. Zhang Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival J. Immunol., February 15, 2003; 170(4): 1846 - 1853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Laub, R. Brecht, M. Dorsch, U. Valey, K. Wenk, and F. Emmrich Anti-Human CD4 Induces Peripheral Tolerance in a Human CD4+, Murine CD4-, HLA-DR+ Advanced Transgenic Mouse Model J. Immunol., September 15, 2002; 169(6): 2947 - 2955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Chiffoleau, G. Beriou, P. Dutartre, C. Usal, J.-P. Soulillou, and M. C. Cuturi Role for Thymic and Splenic Regulatory CD4+ T Cells Induced by Donor Dendritic Cells in Allograft Tolerance by LF15-0195 Treatment J. Immunol., May 15, 2002; 168(10): 5058 - 5069. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Gray, P. Arosio, and P. Hersey Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells Blood, May 1, 2002; 99(9): 3326 - 3334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Spicer, H. Ha, R. A. Boyd, X. Y. He, N. Carter, G. Tran, M. J. Penny, S. J. Hodgkinson, and B. M. Hall IL-4 Therapy Prevents the Development of Proteinuria in Active Heymann Nephritis by Inhibition of Tc1 Cells J. Immunol., October 1, 2001; 167(7): 3725 - 3733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. T. Tran, N. Carter, X. Y. He, T. S. Spicer, K. M. Plain, M. Nicolls, B. M. Hall, and S. J. Hodgkinson Reversal of experimental allergic encephalomyelitis with non-mitogenic, non-depleting anti-CD3 mAb therapy with a preferential effect on Th1 cells that is augmented by IL-4 Int. Immunol., September 1, 2001; 13(9): 1109 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hara, C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell, P. J. Morris, F. Powrie, and K. J. Wood IL-10 Is Required for Regulatory T Cells to Mediate Tolerance to Alloantigens In Vivo J. Immunol., March 15, 2001; 166(6): 3789 - 3796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Nicolls, M. Coulombe, H. Yang, A. Bolwerk, and R. G. Gill Anti-LFA-1 Therapy Induces Long-Term Islet Allograft Acceptance in the Absence of IFN-{gamma} or IL-4 J. Immunol., April 1, 2000; 164(7): 3627 - 3634. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, p <
0.0005 compared with tolerant allografts. Horizontal axis lists
cytokine mRNA assayed. CYT, cytolysin; GRA-A, granzyme A. Vertical axis
gives cycle at which specific PCR product was first detected. Results
are presented as the median ± range. A lower cycle number of
detection indicates a higher concentration of specific mRNA in the
starting sample. Each five-cycle difference represents a 10- to 32-fold
difference in mRNA. All starting samples were standardized for
concentration of RNA and had comparable levels of GAPDH.
