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,
*
Laboratory of Experimental Immunology and
Department of Histopathology, Université Libre de Bruxelles, Brussels, Belgium;
Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, Paris, France; and
Laboratoire d’Immunologie, Institut Curie, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-chain-/- double-mutant H-2k male or
female mice. However, skin from H-2k males, but not from
H-2k females, is acutely rejected by recombinase-activating
gene-2-/- transgenic female recipients. In vitro, Marylin
spleen cells primed by H-2k skin grafting proliferated and
secreted both IL-4 and IFN- in response to H-2k male
stimulators. However, the removal of H-2b APC from the
responding population abolished the response. Taken together, these
results show that the indirect recognition that triggers rejection in
this model is due to the recognition of H-Y Ag shed from
H-2k male allograft and presented by the recipient’s own
I-Ab APC to transgenic T cells. This study demonstrates
unequivocally the capacity of naive CD4+ T cells to promote
the rejection of allografts through mechanisms that involve indirect
destruction of grafted tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ample evidence indicates that indirect allorecognition occurs during allograft rejection. The indirect pathway was put forward as a hypothesis to explain the Ag specificity of the CD4+ T cells responsible for the rejection of MHC class II Ag-deficient allografts (6). Several studies in humans, rats, and mice have revealed the presence of alloreactive CD4+ T cells specific to alloantigens presented as peptide fragments in association with recipient MHC molecules during allograft rejection (7, 8, 9, 10, 11). Moreover, it has been reported that intrathymic administration of allogeneic peptides that are known to stimulate self-restricted alloreactive T cell clones can prolong the survival of subsequent allografts, suggesting that indirect presentation is critical to the rejection process (12).
However, while it is clear that T cell clones sensitized to alloantigens through indirect recognition are present during the process of rejection, it has been hard to demonstrate whether they actually promote rejection. The main obstacle resides in the difficulty of finding situations where indirect sensitization represents the only pathway available for activation of alloreactive T cells. The most convincing approaches that have been tried to date involve either the transplantation of MHC class II molecule-deficient organs or the adoptive transfer of in vitro-derived T cells sensitized by indirect recognition of allopeptides (6, 13). Although elegantly designed, these experiments have major weaknesses. First, they fail to unequivocally show the complete absence of direct recognition of rejected grafts by adoptively transferred T cells (13). Moreover, they are not able to exclude the activity of unusual CD4+ T cells not restricted by conventional MHC class II molecules and capable of mediating allograft rejection (14, 15, 16, 17). Finally, they do not demonstrate whether, in the absence of direct recognition, T cell priming by indirect recognition of allografts is sufficient to bring about rejection. This last point is of particular relevance because memory cells contribute a substantial proportion of the cells involved in primary responses stimulated by direct recognition of donor MHC molecules (18). In contrast, as for any nominal protein Ag, peptides derived either from allogenic MHC molecules or from polymorphic proteins and presented in the context of the recipient’s own MHC molecules stimulate almost exclusively naive T cells.
To establish without ambiguity the capacity of naive CD4+ T cells to reject transplanted foreign tissue through the indirect recognition pathway, we analyzed the rejection of male H-2k skin allografts by recombinase-activating gene-2 (RAG2)-deficient mice expressing an H-Y-specific I-Ab-restricted transgenic (Tg) TCR (Marilyn mice). We show here that in the strict absence of direct recognition, Marilyn CD4+ T cells were not only primed by, but were also able to acutely reject, a male H-2k skin allograft.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Female RAG2-/- Marilyn mice, which are
Tg for a TCR (V
1.1, V
6) specific for an H-Y peptide
(NAGFNSNRANSSRSS) (19) presented by
I-Ab, have been previously described
(20) and were used as recipients of skin grafts. Male and
female C3H (H-2k) and C57BL/6 (B6)
(H-2b) mice were obtained from Harlan Netherlands
(Horst, The Netherlands). Male and female
H-2k or H-2b,
RAG2-/- common -chain
(c)-/- double-deficient mice
were obtained by crossing RAG2-/- B6 (N9 to B6
from the CDTA, Orléans, France) with B10.BR and then with
RAG2-/-
c-/- (N4 to B6 obtained from J.
Di Santo, Paris, France) and were bred at the animal facility of Necker
Hospital (Paris, France).
Adoptive transfer and flow cytometry
TCR Tg CD4+ T cells were purified using
anti-CD4 magnetic beads and the VarioMacs system from Miltenyi
Biotech (Paris, France). Highly purified (>95%) TCR Tg
CD4+ T cells (1 x
106) were injected i.v. into male and female
RAG2-/-
c-/- H-2k
recipients. Seven days later spleen cells were harvested from
adoptively transferred animals, counted, and prepared for analysis by
flow cytometry. APC-anti-CD4 (clone GK1.5), PE-anti-V6
(clone RR4-7), biotin anti-CD62L (clone MEL-14), and
FITC-anti-CD44 (clone IM7) mAbs were purchased from BD PharMingen
(Meylan, France). Streptavidin TriColor was obtained from Caltag (Tebu,
France). Flow cytometry was performed on a FACSCalibur (Becton
Dickinson, Meylan, France).
Skin graft and histology
Female recipients were anesthetized and grafted on the left side of the back with tail skin (70 mm2) from male donors. The grafts were secured using Vaseline gauze and a bandage. Bandages were removed on day 10, and the grafts were then visually scored daily for evidence of rejection. Grafts showing >50% necrosis were considered rejected. Skin grafts were collected from killed mice and stained with hematoxylin and eosin.
Proliferation assay and determination of cytokine secretion
Spleen cells (1 x 105) isolated from
TCR Tg animals at the time of skin graft rejection were mixed with
titrated numbers of irradiated spleen cell stimulators in triplicates
in U-bottom 96-well microplates. [3H]Thymidine
(1 µCi) was added for the final 16 h of a 72-h incubation. In
some experiments, culture supernatants were harvested and studied in a
sandwich ELISA for cytokine production. ELISA kits for mouse IFN-
and IL-4 were supplied by Genzyme (Cambridge, MA). For depletion of MHC
class II Ag-expressing cells in the responding population, spleen cells
were incubated for 30 min at room temperature in culture supernatants
containing rat mAb to mouse MHC class II Ags (clone M5/114) and to
mouse B220 (clone RA3-3A1; American Type Culture Collection, Manassas,
VA). The cells were then submitted to complement lysis (rabbit
complement; Cedarlane Laboratories, Saratoga, Canada) for 45 min at
37°C. Purity was assessed by flow cytometry with FITC-conjugated
anti-I-Ab-specific mAb (clone 25-9-17; BD
PharMingen) specific to an epitope on the class II MHC molecule other
than that recognized by M5/114 Abs. After depletion, <0.3% of cells
expressing MHC class II Ag could be detected in the responding
populations.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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To determine whether CD4+ T cells reject
allografts by the indirect pathway, we first tested the specificity of
the Tg Marilyn CD4+ T cells. Because the Marilyn
clone came from an H-2bxk
F1 female, it seemed unlikely that it would react
against H-2k. However, reactivity against the
higher dose of MHC molecules expressed by a homozygous animal might be
sufficient to activate the Tg T cells. Naive T cells from
RAG2-/- Tg Marilyn mice, which recognize the
male Ag H-Y presented by I-Ab (20),
were cultured with either irradiated H-2b or
H-2k splenocytes. As shown in Fig. 1
a, a vigorous proliferative
response to H-2b male stimulators was observed
without any response toward H-2k-expressing
cells. From these results, one could conclude that Marilyn T cells do
not directly recognize H-2k allogeneic
stimulators. However, because in vitro proliferation is not a sensitive
method to test T cell reactivity, we also set up an in vivo model in
which naive Marilyn T cells were adoptively transferred into
H-2k male hosts. To prevent T cell- or NK
cell-mediated rejection of Marilyn cells, we used hosts deficient for
the common cytokine -chain receptor and Rag
(RAG2-/-
c-/-) (21, 22).
Although these hosts express the H-Y protein, they do not express the
I-Ab restriction element. Thus, their APC should
not be capable of stimulating Marilyn T cells unless the H-Y-specific T
cells can also cross-react against allogeneic
H-2k molecules. When injected into allogeneic
RAG2-/-
c-/- H-2k
male or female hosts (Fig. 1b), purified Marilyn T cells did
not expand for at least 7 days and retained their naive phenotype as
they continued to express low levels of CD44 and high levels of CD62L.
This confirms that they were unable to recognize any Ag presented by
H-2k APC in the adoptive host. In contrast, they
responded well to coinjected syngeneic male spleen cells, undergoing
proliferation 7 days after transfer, with up-regulation of CD44 and
down-regulation of CD62L (Fig. 1b). Thus, antigenic
stimulation of Marilyn CD4+ T cells in the
RAG2-/-
c-/- H-2k
hosts was dependent on the cotransfer of H-Y-bearing
I-Ab APC. Taken together, these results
demonstrate that Marilyn CD4+ T cells do not
directly recognize male or female cells expressing
H-2k alloantigens.
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We next analyzed the capacity of Marilyn mice to reject
H-2k-expressing skin allografts from male donors.
According to the above results, these grafts are not capable of
directly stimulating Marilyn T cells. Therefore, any rejection would be
the result of T cell activation by recipient I-Ab
APC that had processed and presented H-Y Ags shed by the graft. The
survival of skin from H-2k male donors on Marilyn
recipients is compared in Fig. 2 with
that of skin from H-2k female donors. Male
H-2k skin grafts were rejected within 12 days,
whereas female H-2k skin grafts enjoyed
indefinite survival and kept a syngeneic aspect for up to 30 days after
transplantation, when the experiment was terminated. Because our model
involved the transplantation of normal skin onto immunodeficient
recipients, one cannot exclude the possibility that donor-derived T
lymphocytes transferred by the graft could participate in a
graft-vs-host reaction that would initiate the inflammatory processes
leading to allograft rejection (23). To rule out this
possibility, we also transplanted skin allografts isolated from T
cell-deficient RAG2-/- male
H-2k donors onto the Marilyn recipients. As shown
in Fig. 2, the absence of T cells in grafted skin did not modify the
profile of rejection. All RAG2-/-
H-2k-expressing male allografts were acutely
rejected, while female grafts enjoyed long term survival. The histology
of rejected skin allografts revealed the presence of dense cellular
infiltrates in both the derma and epiderma, mainly composed of
macrophage-like cells and lymphocytes, although a few eosinophils were
also noticed (Fig. 3). By contrast,
long-term-surviving female grafts did not show signs of histological
lesions nor did they contain infiltrating lymphoid cells (Fig. 3).
Taken together, these observations show unequivocally that primary
immunization of alloreactive CD4+ T cells by
indirect recognition of graft Ags is sufficient by itself to promote
allograft rejection, therefore demonstrating indirect allorecognition
of grafted tissue as an efficient pathway for the rejection
process.
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The striking features of the rejection of an allograft are the specific destruction of graft elements and the absence of significant damages to host tissues that are close to or in direct contact with the graft. These have led to the widely accepted view that immunologically specific mechanisms of tissue destruction, i.e., CTL activity, Abs, etc., are responsible for the rejection of allografts. Early works on the rejection of mosaic skin from allophenic mice grafted onto one of the parent strains provide direct evidence for the Ag specificity of the effector mechanism of allograft rejection (24, 25, 26). In these grafts only cells that express the target alloantigen are destroyed, whereas cells that are syngeneic to the recipient survive indefinitely. The absence of substantial damage to bystander syngeneic cells in the graft has fostered the idea that allograft rejection is the result of specific killing of graft cells by humoral or cell-mediated cytotoxicity. However, several observations suggest that nonspecific mechanisms of tissue destruction also participate in graft rejection. First is the observation that the syngeneic elements of allophenic grafts endure some degree of damage during rejection on parental recipients, but unlike the allogeneic compartment of the grafts, they are not fully destroyed, and most of them survive indefinitely (25). A second piece of evidence is the rejection of chimeric skin in which graft parenchyma cells are syngeneic to the recipient and only skin passenger leukocytes, i.e., Langerhans cells, express allogeneic MHC molecules (27). In this setting acute rejection would be the direct result of immunologically nonspecific destruction of graft cells. Our study demonstrates the capacity of immunologically nonspecific effector mechanisms to bring about graft rejection. Because of their genetic deficiency, RAG2-/- TCR Tg recipients do not contain mature B or T cells other than those expressing the TCR transgene and thus cannot develop Ag-specific effector mechanisms of rejection against male H-2k allografts.
Rejection of H-2k skin allograft does not polarize Marilyn CD4+ T cells toward Th1 or Th2 phenotype
We next analyzed the reactivity of Tg Marilyn T cells from mice
that had rejected H-2k male skin allografts. For
MLR, spleen cells were stimulated with irradiated
H-2k or H-2b male or female
stimulators. As depicted in Fig. 4, unpurified Marilyn spleen cells proliferated in response to both
H-2k and H-2b male cells,
while they did not respond to female stimulators. The cells also
specifically secreted both IFN- and IL-4 (Fig. 4). However,
reactivity toward H-2k male cells was completely
abrogated by depletion of MHC class II Ag-expressing cells from the
responding population (Fig. 4). This observation confirms that
Marilyn’s reactivity toward H-2k male cells is
indeed the result of indirect recognition of H-Y presented by
I-Ab and requires H-2b APC
in the assay. The fact that IFN- and IL-4 were secreted in vitro by
Marilyn T cells after stimulation with male cells suggests that both
Th1- and Th2-dependent effector pathways of rejection operate in this
system. Among pathways considered as being Th1 mediated, classical CTL
can be ruled out, because it requires cognate recognition between
effector CD4+ T cells and target graft cells.
However, because keratinocytes, like hepatocytes, are sensitive to
CD95-mediated apoptosis and express CD95L following exposure to
inflammatory cytokines (28), one should not discard a
possible role for Fas-mediated apoptosis of keratinocytes in the
rejection process (29). The presence of numerous
macrophage-like cells in allograft infiltrates suggests a delayed-type
hypersensitivity-like reaction to be the main pathway of rejection. In
contrast, the presence of eosinophilic infiltrates in rejected
allografts supports the idea that a Th2-dependent pathway of rejection
involving IL-5 and eosinophils may be operating in our model
(30). Because we used RAG2-/-
recipients, a role for B cell Abs and classical CTL in the rejection
process can be ruled out.
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It is often assumed that, during rejection, the peptides recognized by indirect pathway CD4+ T cells come mainly from the processing and presentation by recipient APCs of polymorphic moieties derived from donor MHC molecules (31). Our study confirms early findings by Wettstein et al. (32) that rejection by T cells also involve the indirect recognition of peptides from minor transplantation Ags. Thus, minor histocompatibility Ags, such as H-Y Ag and polymorphic molecules, must also be considered as a source of allopeptides for indirect allorecognition in the design of peptide-based immune intervention that would interfere with the rejection process.
The important finding of our study is that the indirect pathway of alloantigen recognition can alone mediate the acute rejection of fully histoincompatible allografts. Because we are using TCR Tg recipients, one could question the relevance of such an observation. Indeed, it could be argued that this situation results from the stimulation of an abnormally high number of indirect pathway T cells and that this would not occur in non-Tg recipients. In animal models, direct pathway T cells have been estimated to represent >90% of the T cell repertoire participating in the process of acute rejection, whereas indirect pathway T cells would include only 1–10% (11). However, one should not forget that the ratio of direct-over-indirect pathway T cells might be profoundly perturbed in clinical situations where sensitization to donor alloantigens has taken place before transplantation (33, 34). Indeed, previous exposure to antigenic peptides has been reported to induce dramatic expansion of Ag-specific oligoclonal T cell populations (35, 36, 37). Thus, in some donor/recipient combinations, indirect pathway T cells may dominate the alloresponse and mediate allograft rejection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michel Y. Braun, Laboratory of Experimental Immunology, Université Libre de Bruxelles, 808 route de Lennik, Brussels B-1070, Belgium.
3 Abbreviations used in this paper: DC, dendritic cells; Tg, transgenic; RAG, recombinase-activating gene; c, common -chain.
Received for publication October 18, 2000. Accepted for publication February 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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+ cells. Immunology 85:531.[Medline]
/
-expressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens. Blood 87:4463. chain (c)-dependent cytokines are essential for naive CD4+ T cell survival but dispensable for antigen driven proliferation and memory responses in vivo. Nat. Immunol. 1:54.[Medline]
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) spleen cells for 72 h. The extent of proliferation was
assessed by [3H]thymidine incorporation for the last
16 h of culture. b, Purified T cells from
RAG2-/- TCR Tg Marilyn mice were injected into allogeneic
RAG2-/- 
)
or male (n = 6; 
) or male
(n = 5; 
) RAG2-/- H-2k
mice were transplanted onto Marilyn TCR Tg recipients. The results
represent two pooled independent experiments.
