By
From the * Thymus Biology Unit, The Walter and Eliza Hall Institute of Medical Research, Royal
Melbourne Hospital, Parkville 3050, Victoria, Australia; and the Monash Medical School, Alfred
Hospital, Prahran 3181, Victoria, Australia
In this report, we show that cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8+ T cells. We had previously shown that transfer of ovalbumin
(OVA)-specific CD8+ T cells (OT-I cells) into rat insulin promoter-membrane-bound form of
OVA transgenic mice, which express the model autoantigen OVA in the proximal tubular cells
of the kidneys, the cells of the pancreas, the thymus, and the testis of male mice, led to the activation of OT-I cells in the draining lymph nodes. This was due to class I-restricted cross-presentation of exogenous OVA on a bone marrow-derived antigen presenting cell (APC) population. Here, we show that adoptively transferred or thymically derived OT-I cells activated by
cross-presentation are deleted from the peripheral pool of recirculating lymphocytes. Such deletion only required antigen recognition on a bone marrow-derived population, suggesting
that cells of the professional APC class may be tolerogenic under these circumstances. Our results provide a mechanism by which the immune system can induce CD8+ T cell tolerance to
autoantigens that are expressed outside the recirculation pathway of naive T cells.
Several mechanisms play a role in tolerance induction to
extra thymic self-antigens. For class I-restricted CD8+
T cells, ignorance, anergy, and deletion can operate to render an animal tolerant to antigen expressed in peripheral
tissues (1). However, the current dogma provides an interesting dilemma with regard to our understanding of how
tolerance is achieved. Anergy and deletion both require interaction of T cells with the self-antigen, and naive T cells
are thought not to recirculate through nonlymphoid tissue
(15). Thus, those CD8+ T cells specific for antigens expressed only in nonlymphoid tissues, e.g., in the To date, many studies examining peripheral tolerance of
CD8+ T cells have used MHC molecules as their model
autoantigens (5, 14, 16). These molecules are seen only
in an unprocessed form, and therefore only on those cells
that themselves express the autoantigen. Although such
studies have contributed to our understanding of the fate of
autoreactive CD8+ T cells, they have not allowed for the
possible effects of processing and presentation of tissue antigens by professional APCs.
Peptide antigens presented by MHC class I molecules are
generally thought to be derived from intracellularly synthesized proteins (17). However, exogenous antigens can
also be presented by class I MHC molecules under certain
circumstances (20) and the induction of CTL via this
exogenous pathway for class I-restricted presentation has
been referred to as cross-priming. We have recently shown that such presentation, when applied to self-antigens, can lead to the activation of autoreactive CD8+ T cells (27). These
studies used the rat insulin promoter (RIP)1-mOVA transgenic mouse model, where a membrane-bound form of
ovalbumin (mOVA) was expressed by pancreatic Mice.
All mice were bred and maintained at the Walter and
Eliza Hall Institute of Medical Research. For all experiments, female mice between 8 and 16 wk of age were used. RIP-mOVA
and OT-I transgenic mice were generated and screened as previously described (27, 28).
Adult Thymectomized, Thymus-grafted, Bone Marrow Chimeras.
Adult thymectomized, thymus-grafted, bone marrow chimeric RIP-mOVA mice (TG-RIP mice) and nontransgenic mice (TG-nontransgenic mice) were generated as described (9, 14). In brief, 2-6 wk after adult thymectomy RIP-mOVA mice and their nontransgenic littermates were lethally irradiated with 900 cGy and reconstituted with T cell-depleted bone marrow from OT-I mice. The
next day, radioresistant T cells were depleted with 100 µl of T24
(anti-Thy-1) ascites intraperitoneally. 1-2 wk after irradiation, mice
were grafted with a sex-matched 1,500 cGy irradiated thymus
graft from a nontransgenic newborn B6 mouse under the kidney
capsule. This approach ensured that OT-I cells were not deleted
intrathymically due to aberrant expression of OVA in this tissue.
Preparation of OT-I Cells for Adoptive Transfer.
OT-I RAG-1 5,6-Carboxy-Succinimidyl-Fluoresceine-Ester Labeling of OT-I
Cells.
5,6-carboxy-succinimidyl-fluoresceine-ester (CFSE) labeling was performed as previously described (29). OT-I cells
were resuspended in PBS containing 0.1% BSA (Sigma Chem.
Co., St. Louis, MO) at 10 × 106 cells/ml. For fluorescence labeling, 2 µl of a CFSE (Molecular Probes, Eugene, Oregon) stock
solution (5 mM in DMSO) were incubated with 10 × 106 cells
for 10 min at 37°C.
FACS® Analysis.
LN or spleen cells were stained for three-color FACS® analysis as described (10), using the following mAbs:
PE-conjugated anti-CD8 (YTS 169.4) was from Caltag (San Francisco, CA). Biotinylated anti-CD69 (H1.SF3) and PE-labeled anti-
L-selectin (Mel-14) were from PharMingen (San Diego, CA). Anti-V cells of
the pancreas, should not be susceptible to these forms of
tolerance. This leaves ignorance as the only mechanism for
avoiding autoimmunity to such antigens, a somewhat unsatisfactory situation because activated CD8+ T cells have
wider recirculation pathways than naive cells (15), and can
potentially cause autoimmune damage in tissues previously ignored (3, 4, 10). Consequently, we might expect autoimmunity to be more prevalent, or that additional tolerogenic
mechanisms exist.
cells, kidney proximal tubular cells, the thymus, and in the testis of
male mice. Transgenic OVA-specific CD8+ T cells (OT-I
cells) adoptively transferred into RIP-mOVA mice were
activated in the lymph nodes draining OVA-expressing tissues. This activation was due to class I-restricted presentation
of exogenously derived OVA on a bone marrow-derived
APC population. Here, we investigate the fate of autoreactive CD8+ T cells activated by this cross-presentation pathway and provide evidence that these cells are deleted.
/
cells were prepared from LN and spleens of transgenic mice as described (27). In brief, erythrocytes and macrophages were removed by treatment with the anti-heat stable antigen mAb J11d
and complement. OT-I cells from RAG-1
/
mice were of a naive phenotype (CD44lo, CD69
, IL-2R
). 0.25-6 × 106 cells
were injected intravenously into recipient mice.
2 TCR (B20.1) and anti-V
5.1/2 TCR (MR9-4) mAbs (27)
were conjugated to biotin or to FITC. Biotin-labeled Abs were
detected with Streptavidin (SAVP)-Tricolor (Caltag). Analysis was
performed on a FACScan® using Lysis II software. Live gates
were set on lymphocytes by forward and side scatter profiles.
10,000-20,000 live cells were collected for analysis. OT-I donor
cells in the LNs from recipient mice were identified by staining
for V
2+ V
5+ CD8+ cells.
Histology. Tissues were fixed in Bouin's solution and paraffin sections were stained with hematoxylin and eosin using standard methods.
RIP-mOVA mice express a membrane-bound form of OVA in the cells of the pancreas,
the proximal tubular cells of the kidney, the thymus, and in
the testis of male mice (27). When OVA-specific CD8+ T
cells from the OT-I transgenic line (OT-I cells) were transferred into RIP-mOVA mice, they were activated in the
draining lymph nodes of OVA-expressing tissues (27). In
this report, we have used a novel and more sensitive
method for the identification of proliferating cells in vivo
(29). OT-I cells were labeled with the fluorescent dye
CFSE and then transferred into RIP-mOVA mice. When
CFSE-labeled cells divide, the two daughter cells receive approximately half of the original fluorescence, and their
progeny a quarter, and so on. Thus, a cell that has divided n
times will exhibit a 2n-fold reduced fluorescence intensity.
Therefore, on a FACS® histogram, separate peaks appear
for cells that have divided 1-8 times. After nine cell cycles,
the fluorescent dye is diluted to background intensity. Fig.
1 shows the CFSE profiles of 5 × 106 OT-I cells transferred into RIP-mOVA mice 43 h earlier. Multiple peaks
are seen only in the renal (Fig. 1 A) and pancreatic (Fig. 1
B) lymph nodes, confirming that OT-I cells were activated
and proliferated only in these sites.
Using the above technique, divided cells were first apparent at 25 h after transfer (data not shown). After 43 h,
some OT-I cells had divided four times (Fig. 1), six times
within 52 h (data not shown), greater than eight times
within 68 h (Fig. 2). Therefore, one cell cycle required
~4.5 h in vivo.
These results indicate that OT-I cells were able to respond to antigen presented in the lymph nodes that drained
OVA-expressing tissues. Previously, we showed that in the
absence of a bone marrow-derived APC capable of presenting OVA to OT-I cells, no proliferation was observed
(27). To determine whether OVA presentation by such
bone marrow-derived APCs alone was sufficient to induce
OT-I cell proliferation, we took advantage of bm1 mice,
which express a mutation in the Kb molecule such that
Kbm1 cannot present OVA to OT-I cells (32). RIP-mOVA
mice, which were crossed onto the bm1 haplotype (RIP-
mOVA.bm1), were lethally irradiated and reconstituted with
B6 bone marrow (B6 RIP-mOVA.bm1). In these chimeric mice, Kb is expressed by bone marrow-derived cells
but not by peripheral tissue cells such as islet
cells or kidney proximal tubular cells. 5 × 106 CFSE-labeled OT-I
cells were adoptively transferred into the chimeric RIP-
mOVA mice and, 3 d later, lymphoid tissues were analyzed
by flow cytometry (Fig. 2). Proliferation of OT-I cells was
observed in the renal (Fig. 2 C) and pancreatic (data not shown) nodes, but not in the inguinal lymph nodes of
B6
RIP-mOVA.bm1 chimeras (Fig. 2 G). This result
showed that antigen presentation by bone marrow-derived
cells was sufficient to induce proliferation of OT-I cells.
The proliferation was not as intense as in normal RIP-
mOVA mice (see Fig. 1), but was comparable to that seen
in B6
RIP-mOVA.B6 control chimeras that were entirely of the B6 haplotype (Fig. 2, A and E). This implied
that the less vigorous response seen in chimeric mice may
be the result of irradiation. As previously reported (27),
OT-I cells were not activated when the bone marrow compartment was of the bm1 haplotype (bm1
RIP-mOVA.B6 or bm1
mOVA.bm1), indicating that antigen presentation by a bone marrow-derived cell was not only sufficient,
but also essential for OT-I activation.
The above results indicate that a
bone marrow-derived APC was capable of processing and
presenting antigens expressed by peripheral tissues for activation of autoreactive CD8+ T cells. To determine how
the immune system normally copes with such autoreactive
cells, we examined the ultimate fate of these cells. To detect adoptively transferred OT-I cells in unirradiated recipients several weeks after transfer, it was necessary to inject at least 5 × 106 cells. However, under these circumstances
OT-I cells infiltrated the pancreatic islets after day 3, and
caused diabetes in 100% of 16 RIP-mOVA mice by day 9 (data not shown). Smaller numbers of cells, e.g., 0.25 × 106 cells, did not cause diabetes in 25 recipients, but detection of these few cells was not possible several weeks after
transfer, even in nontransgenic controls. Presumably, OT-I
cells were activated after transfer in both cases, but only the
larger dose caused sufficient destruction to result in diabetes. To avoid the problem of cell destruction, we transferred 6 × 106 OT-I cells into B6
RIP-mOVA.bm1 chimeric mice, in which OT-I cells could recognize antigen
on the cross-presenting bone marrow-derived APCs, but
could not interact with OVA-expressing peripheral tissues of the bm1 haplotype. After 8 wk, far fewer OT-I cells
were recovered from the lymphatic tissues of B6
RIP-
mOVA.bm1 mice than from nontransgenic B6
bm1 mice
(Fig. 3). These data suggest that OT-I cells were deleted after recognizing exogenously processed OVA on bone marrow-derived APC in the draining lymph nodes of OVA-expressing tissue.
Deletion of Constitutively Produced OT-I Cells in the Periphery of RIP-mOVA Mice.
The adoptive transfer of 5 × 106 OT-I cells contrasts with the normal situation where small numbers of newly matured cells enter the periphery from the thymus each day. We reasoned that diabetes may have occurred because the normal tolerogenic mechanisms were unable to cope with such a large number of injected T cells.
To create a more physiological situation where OVA-specific CD8+ T cells would be generated continuously in the thymus, RIP-mOVA mice were manipulated to ensure that OVA could not be expressed in this compartment. This was achieved by thymectomizing RIP-mOVA mice and then grafting them with a nontransgenic B6 thymus. Such mice were then lethally irradiated and reconstituted with bone marrow from OT-I mice, and designated thymus-grafted RIP-mOVA mice (TG-RIP mice). This approach has been successfully used to exclude the effect of aberrant thymic antigen expression in other models (10, 14).
In contrast with the RIP-mOVA mice given large numbers of OT-I cells, which became diabetic within 9 d, only
1 of 12 TG-RIP mice developed the disease when followed
for >116 d. Analysis of the thymus grafts 4 mo after implantation showed that OT-I cells (CD8+CD4V
2+ cells)
were able to mature in TG-RIP mice (Fig. 4). The proportion of mature OT-I cells in the thymus was equivalent to
that of nontransgenic controls (Fig. 4, A and D), supporting
the view that the thymic deletion reported earlier for the
double-transgenic mice (27) was the result of aberrant thymic expression of mOVA.
Because OT-I cells matured in the thymus of TG-RIP mice, we could examine their fate after release into the peripheral immune system. Flow cytometric analysis of the lymph node populations of TG-RIP mice showed a significant reduction in the proportion of these cells relative to that seen in TG-nontransgenic mice (4.9 ± 2.4% versus 25.1 ± 8.2%, n = 8) (Fig. 4, C and F). Calculation of the total number of OT-I cells in the spleen and lymph node populations indicated that there was approximately a fivefold reduction of these cells in TG-RIP mice (2.28 ± 1.21 × 106 versus 12.06 ± 2.10 × 106; n = 8). The remaining OT-I cells in TG-RIP mice proliferated in vivo after restimulation with antigen, demonstrating that they were not anergic (data not shown). These data strongly suggest that OVA-specific CD8+ T cells were lost and probably deleted once they entered the periphery.
Consistent with an active deletional process occurring in
these mice, OT-I cells from the peripheral lymphatic tissues of TG-RIP mice expressed elevated levels of the activation marker CD69 (Fig. 5, A-C) and decreased levels of
L-selectin (Fig. 5, D-F) relative to that seen in TG-nontransgenic control mice. The proportion of activated OT-I
cells was even higher in the lymph nodes draining OVA-expressing tissues (Fig. 5, C and F), suggesting that activation of OT-I cells in TG-RIP mice also occurred in these
draining lymph nodes, presumably by the same cross-presentation mechanism that activates adoptively transferred
OT-I cells.
To determine the fate of those few activated OT-I cells
remaining in the periphery of TG-RIP mice, the thymus
grafts were removed 3 mo after implantation to stop further
T cell production. The proportion of OT-I cells was then
examined in the blood at various later timepoints. This revealed a continuous decline in the proportion of OT-I cells
in TG-RIP mice (Fig. 6), consistent with the idea that a
continuous deletion process was in operation. These few
remaining cells were also able to proliferate upon restimulation with antigen in vivo (data not shown).
There are now numerous reports showing that cross-presentation of exogenous antigen can prime class I-restricted CTL responses (33). It has also been shown to induce tolerance in the thymus (23). Here, we show that cross-presentation can induce peripheral tolerance that appears to operate via a deletional process.
Although our data strongly suggest that OT-I cells were deleted in TG-RIP mice, an alternative possibility is that these cells had relocated to extralymphoid sites. However, because few OT-I cells were seen in nonlymphatic tissues, apart from small numbers of cells in the pancreatic islets (data not shown), this possibility seems remote. It should be emphasized that the mild islet infiltration observed is unlikely to account for the loss of ~107 cells from the secondary lymphoid organs of the TG-RIP mice.
Deletion has been reported as a likely mechanism of extrathymic tolerance for several introduced antigens, including superantigens (34, 35), viruses (36), soluble peptides (37, 38), and protein (39), and for some self-antigens in transgenic models (8, 14, 16, 40). The general belief is that this form of tolerance results from exhaustive differentiation (34). T cells are stimulated so extensively by antigen that they proceed to end-stage effectors with limited lifespan. Such a mechanism is consistent with the observed activation and proliferation that precedes deletion in the RIP-mOVA model.
Our findings provide a pathway whereby CD8+ T cells
can be tolerized to self-antigens expressed in tissues outside the
normal recirculation pathway for naive T cells. As long as the
antigen can gain access to the exogenous cross-presentation
pathway, host CD8+ T cells should be stimulated to die
eventually. Thus, as newly derived autoreactive CD8+ T cells
enter the peripheral pool, they will encounter their autoantigen on the cross-presenting APC in lymph nodes that drain the appropriate tissues. As a result, activation will follow
and lead to deletion, thus limiting the accumulation of ignorant autoreactive cells in secondary lymphoid compartments. This model is at odds with the previously reported
induction of diabetes in virus-primed transgenic mice expressing viral antigens in the islet cells (3, 4), which suggests that ignorant naive CD8+ T cells remained in the peripheral circulation. However, the type and concentration
of the antigen and the affinity of the TCR in the responding T cell population are likely to affect the efficacy of cross-presentation leading to deletion. In support of this conclusion, we have preliminary evidence using newly generated transgenic lines that the level of antigen expressed affects the extent of cross-presentation (our unpublished observations).
In addition, data obtained using transgenic mice expressing
hemagglutinin (HA) under the control of the rat insulin
promoter (RIP-HA mice), support the idea that antigens
expressed in the islets are not always ignored but can activate CD8+ T cells leading to tolerance induction. RIP-HA
mice crossed to TCR transgenic mice, which produced large
numbers of class I-restricted HA-specific T cells, became
diabetic (41), indicating that HA-specific CD8+ T cells entered the periphery of RIP-HA mice, where they were activated by islet antigens, perhaps by cross-presentation.
Despite this observation, RIP-HA single-transgenic mice
showed HA-specific CTL tolerance (11), suggesting that the
activation process seen in double-transgenic mice may lead
to tolerance induction when the precursor frequency is
low, as in a normal T cell repertoire.
It is not clear why this form of priming should lead to loss of activated cells when most other described cases of cross-priming result in immunization. Perhaps it relates to the continuous presence of the priming antigen, which provides repeated stimuli to the responding population to the point of exhaustion. Alternatively, because there is specific tolerance to OVA in the CD4+ T cell compartment in our model (our unpublished observations), deletion of CD8+ cells may relate to a lack of CD4+ T cell help, which appears to be necessary for the induction of some CD8+ T cell responses (42). Thus, when CD8+ T cells were confronted with antigen in the absence of CD4+ T cells, only a transient response followed after which all the antigen-specific T cells died (45).
Deletion of OT-I cells in TG-RIP mice is consistent
with a model in which newly matured OT-I cells enter the
periphery of RIP-mOVA mice, recirculate until they come
into contact with antigen in the draining lymph nodes of
OVA-expressing tissues, and are then activated and deleted.
Such activation-induced deletion could occur in one of two
ways: either directly as a result of activation on the bone
marrow-derived cross-presenting APC, or indirectly because only activated OT-I cells are able to recirculate
through nonlymphoid tissues where they can encounter
OVA-expressing tissues and there be deleted. The observed
deletion of OT-I cells adoptively transferred into B6 RIP-
mOVA.bm1 mice indicates that secondary encounter with
antigen on peripheral tissues is not essential for deletion to
occur. However, it is important to state that although presentation of OVA on the bone marrow-derived compartment was sufficient to lead to deletion, the additional ability to encounter antigen on peripheral tissue may enhance
deletion. This will be examined in future studies.
Our results provide evidence for an extrathymic mechanism capable of inducing the loss of CD8+ T cell responding to self-antigens expressed in tissues outside the lymphoid compartment. Because such tissues are normally not directly accessible to naive CD8+ T cells, the absence of this deletion mechanism would allow accumulation of naive autoreactive CD8+ T cells. These could be primed to effector CTL with wider recirculation pathways after a strong environmental stimulus, thus leading to autoimmunity. We speculate that the continual activation and deletion of small numbers of autoreactive CD8+ T cells by cross-presentation will not result in significant autoimmune damage.
Address correspondence to Jacques F.A.P. Miller, Thymus Biology Unit, The Walter and Eliza Hall Institute, Post Office Royal Melbourne Hospital, Parkville 3050, Victoria Australia. Phone: 61-3-9345-2555; FAX: 61-3-9347-0852; E-mail: miller{at}wehi.edu.au. The current address for Hiroshi Kosaka is the Department of Dermatology, Osaka University Medical School, 2-2 Yamada Oka, Suita, Osaka 565, Japan.
Received for publication 4 March 1997 and in revised form 5 May 1997.
C. Kurts is supported by a fellowship from the Deutsche Forschungsgemeinschaft (Grant Ku1063/1-I). This work was funded by National Institutes of Health grant AI-29385 and grants from the National Health and Medical Research Council of Australia and the Australian Research Council.We thank P. Hodgkin for help with the CFSE labeling technique; J. Falso, T. Banjanin, F. Karamalis, and P. Nathan for their technical assistance.
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CD4+8![]() |