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Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T
cells (DNTC) in the periphery of TCR transgenic mice are resistant to
clonal deletion in cognate Ag-expressing (Ag+) mice.
Previously, we have characterized DNTC populations bearing the
alloreactive 2C TCR in Ag-free (Ag-) and Ag+
mice. Despite appearing functionally anergic when challenged with
cognate Ag in vitro, Ag-experienced DNTC exhibit markers of
activation/memory, a lowered threshold of activation, ex vivo cytolytic
activity, and the ability to rapidly secrete IFN-
. Remarkably, these
memory-like DNTC also possess potent immunoregulatory properties,
competing effectively for bystander-produced IL-2 and suppressing
autoreactive CD8+ T cell proliferation via a
Fas/FasL-dependent cytolytic mechanism. The fact that DNTC recovered
from Ag+ mice possess markers and attributes characteristic
of naive CD8+ T cells that have undergone
homeostasis-induced proliferation suggested that they may be derived
from a similar peripheral expansion process. Naive DNTC adoptively
transferred into Ag-bearing hosts rapidly acquire markers and
functional attributes of DNTC that have continually developed in the
presence of Ag. Thus, the peripheral selection and maintenance of such
autoreactive cells may serve to negatively regulate potential
autoimmune T cell responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to regulating their responses to cognate Ag, T cells also
function in maintaining the size of the peripheral T cell pool
(5). For example, the adoptive transfer of naive
CD4+ or CD8+ T cells into a
lymphopenic environment results in them abandoning their quiescent
state and undergoing extensive proliferation. Such homeostasis-driven
expansion does not result from stimulation with cognate Ag, rather it
requires low-affinity interactions with self peptides/MHC complexes
(5). However, neither costimulation through CD28 nor
accessibility to IL-2 appears critical for this process
(6). Although the slow proliferation driven by homeostatic
forces does not result in the up-regulation of acute activation markers
like CD69 and CD25 or effector cell formation, these dividing cells do
acquire markers and characteristics of memory T cells
(6, 7, 8). In contrast to naive T cells,
CD8+ T cells derived from homeostatic expansion
mount accelerated responses to cognate Ag, possess significant CTL
activity, and produce IFN- without a requirement for prior
stimulation. Conversion of naive cells into memory ones through
lymphopenia-induced homeostatic proliferation may account for the
increased frequency of memory/activated T cells in lymphopenic
individuals such as patients suffering with AIDS or immunosuppressive
regimens (9).
The peripheral lymphoid organs of TCR transgenic mice contain
significant numbers of
CD4-CD8- T cells that
express the transgenic TCR
(DNTC3; Ref.
10). Unlike conventional cells, DNTC from TCR transgenic
mice do not express endogenous TCR genes (11), they
develop independently of class I MHC molecules (12, 13),
and they are resistant to clonal deletion in cognate Ag-expressing mice
(10, 12). We have previously characterized mature DNTC
bearing the alloreactive 2C TCR in cognate Ag-free
(H-2b) and Ag-expressing
(H-2d) mice (14). The 2C TCR
recognizes the naturally occurring peptides p2Ca and QL9 (both derived
from the same ubiquitous protein) in the context of
Ld (15, 16), and it is positively
selected by Kb MHC class I molecules
(17). To examine the effects of chronic exposure to
physiological levels of cognate antigenic ligand, we performed
functional analyses on DNTC from Ag-free (Ag-)
and Ag+ mice. Initial comparisons of these two
populations revealed that DNTC from Ag+ mice
appeared functionally anergic as they hypoproliferate and produce
little or no IL-2 in response to Ag stimulation (14).
However, they differ from conventional in vivo anergized T cells
(18, 19) because this type of anergy is reversible by the
addition of exogenous IL-2. Interestingly, DNTC from
Ag+ mice also possess a lowered activation
threshold as they respond to a low-affinity ligand by up-regulating
CD25 and CD69 (20). By contrast, DNTC from
Ag- mice are refractory to stimulation by the
low-affinity ligand. In this study, we demonstrate that this
autoreactive population has a number of features in common with naive
CD8+ T cells that have undergone homeostatic
proliferation and conversion into memory-like T cells. Furthermore,
DNTC from Ag+ mice can sequester
bystander-produced cytokine to drive their proliferation and cytolytic
effector function. Importantly, these DNTC could suppress autoreactive
CD8+ T cell proliferation by using a
Fas/FasL-dependent cytolytic mechanism. Collectively, our data suggest
a novel mechanism for the preservation of self-tolerance. This
mechanism is dependent on the differentiation of DNTC into regulatory
effector cells following a self Ag-driven peripheral expansion process
that resembles lymphopenia-induced CD8+ T cell
homeostatic proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeders for the H-2b 2C TCR transgenic mice were kindly provided by Dr. D. Y. Loh (Washington University, St. Louis, MO). The 2C TCR transgenic mice were bred onto the C57BL/6 (H-2b) background. H-2b/d 2C were F1 mice generated by mating H-2b 2C mice with DBA/2 (H-2d). C57BL/6-lpr/lpr mice were obtained from The Jackson Laboratory (Bar Harbor, ME). H-2b 2C mice were also crossed onto a Fas-deficient (lpr) background. Mice were housed under pathogen-free conditions in the Department of Microbiology and Immunology’s animal facility at the University of British Columbia (Vancouver, Canada). All studies followed the guidelines set by the university’s Animal Care Committee and the Canadian Council on Animal Care.
CFSE labeling
DNTC (1 x 107/ml) were labeled with 1 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 10 min at room temperature. After stopping the reaction with the addition of an equal volume of FCS, cells were washed four times with complete media.
APCs and peptides
The peptide transporter-deficient cell lines T2-Ld and T2-Kb were created by transfection of the human (T x B) hybridoma T2 with either Ld or Kb mouse MHC class I molecules (21). The p2Ca (LSPFPFDL; Ref. 22) and pOVA (SIINFEKL; Refs. 16, 23, 24) peptides were synthesized at the University of British Columbia’s Nucleic Acid-Protein Service Unit.
Proliferation assays
DNTC were purified as previously described (14). Ten thousand DNTC were stimulated with 5 x 104 mitomycin C-treated T2-Ld cells loaded with 1 µM p2Ca peptide. DNTC proliferation was compared with p2Ca/APCs alone or with 10 ng/ml rIL-2 or rIL-15 (R&D Systems, Minneapolis, MN). Cells were cultured in a 200-µl volume in a U-bottom 96-well plate. After 72 h, cultures were pulsed with 1 µCi of [3H]thymidine for 8 h to assess proliferation.
RT-PCR
RNA was purified from Ag-stimulated DNTC. Briefly, 1 x
105 DNTC were incubated with 5 x
104 Ld-expressing APCs
(T2-Ld) loaded with 1 µM p2Ca peptide in a
U-bottom 96-well plate for 8 h. Cells from 48 wells were pooled
per sample, and RNA was prepared using the RNeasy kit (Qiagen,
Valencia, CA) according to manufacturer’s instructions. Following
reverse transcription, cDNA samples were normalized for hypoxanthine
phosphoribosyltransferase content using competitive-quantitative
PCR before analyzing IL-2, TNF-, and TGF- expression. The primers
used to detect expression of these cytokines and the competitor, pPQRS
plasmid, have been described previously (25). To detect
IL-15 expression, primers specific to two different exons were used to
amplify a 290-bp fragment by RT-PCR. The sequences of the primers read
as follows: 5'-ACCACTTTATACACTGACAGTGAC-3' and
5'-CTCGCATGCAGTCAGGACGTGTTG-3'. Digitized images of
ethidium-stained agarose gels were inverted in Adobe Photoshop (Adobe
Systems, Mountain View, CA).
Flow cytometry
Single cell suspensions from lymph nodes (LNs) were
subjected to RBC lysis by ammonium chloride. All incubations were done
in FACS buffer (2% FCS in PBS) on ice for 15 min. All Ab reagents were
purchased from BD PharMingen (San Diego, CA) except for anti-mouse
CD8-Tricolor, which was purchased from Caltag Laboratories (Burlingame,
CA), and 1B11 Ab (26), which was purchased from H. J.
Ziltener (University of British Columbia). Data was acquired using a
FACScan and it was analyzed by CellQuest software (BD Biosciences). For
intracellular IFN- detection, LN cells (5 x
105) were stimulated with
T2-Ld cells loaded with 1 µM p2Ca for 8 h.
To block cytokine secretion, Golgi Plug (BD PharMingen)
was added 3 h into the incubation. At the finish of the
stimulation, the cells were labeled with cell surface markers before
fixing with 2% paraformaldehyde in PBS. After permeabilization with
0.3% saponin in PBS, cells were stained intracellularly, washed, and
analyzed by flow cytometry.
Coculture to monitor bystander proliferation
Fifty thousand labeled DNTC were preactivated for 24 h with Ag (T2-Ld plus 1 µM p2Ca) before the addition of either media alone or 5 x 104 2C CD8+ T cells. After culture for an additional 2 days, DNTC proliferation was tracked by flow cytometry.
Coculture to monitor DNTC suppressor activity
Fifty thousand unlabeled DNTC were preactivated with cognate Ag for 24 h before the addition of 5 x 104 CFSE-labeled 2C CD8+ T cells. Labeled CD8+ T cells were either added to wells containing activated DNTC or Ag alone. Proliferation of CD8+ T cells was measured by flow cytometry 3 days following their stimulation.
Direct ex vivo CTL assays
DNTC were purified from peripheral LN. TAP-deficient cells (T2-Kb or T2-Ld) expressing either Kb or Ld were 51Cr labeled. Washed targets (1 x 104) were mixed with various ratios of effectors and were incubated in 200 µl of media in 96-well V-bottom plates. Assays were done in the presence and absence of 1 µM p2Ca peptide. After 5 h of incubation, supernatants were collected and counted. The spontaneous release varied between 10 and 15% of the maximum release counts. All assays were performed in triplicate. Percent maximum release was calculated as 100% x [cpm (experimental well) - cpm (spontaneous release)]/[cpm (maximum release) - cpm (spontaneous release)].
CTL assays with ex vivo activated effectors
DNTC or 2C CD8+ T cell effectors were generated by a 3-day stimulation with Ag and IL-2. To produce targets, CD8+ T cells were positively selected by magnetic separation using the MiniMACS system (Miltenyi Biotec, Auburn, CA) according to manufacturer’s specifications. After activating the purified CD8+ T cells with ConA and IL-2 for 40 h, blasts were labeled with 51Cr for 1 h at 37°C. Washed targets (1 x 104) were incubated with 1 x 105 effectors for 5 h before collection of supernatants for counting. The pOVA and p2Ca peptides were used at a final concentration of 1 µM. Fas/FasL interactions were blocked by the addition of 10 µg/ml recombinant Fas-Fc fusion protein (Immunex, Seattle, WA).
Adoptive transfers
Naive DNTC and CD8+ T cells were recovered from the LNs of H-2b 2C TCR transgenic mice. Ag-experienced DNTC were obtained from H-2b/d 2C TCR transgenic mice. Purified T cell populations were labeled with CFSE, and 1 x 106 cells were transferred per mouse via tail vein injections. C57BL/6 (B6) and BDF1 (B6 x DBA/2 F1) recipient animals were subjected to 600 rad of gamma irradiation 5 h before cell transfer. Proliferation assays were set up as described above except that 2 x 103 1B2+ cells were added per well.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A comparison of the proportion of DNTC in
Ag- and Ag+ mice revealed
a 2-fold increase in DNTC in the peripheral lymphoid organs of
Ag+ mice (Fig. 1
A). Associated with this
increased incidence, there is a 
50% increase in the DNTC yield from
Ag+ mice. Despite the persistence of autoreactive
DNTC, Ag+ mice fail to display overt signs of
autoimmunity. We have previously noted that tolerance induction of
these self-reactive cells may involve T cell anergy (14).
Consistent with T cell anergy, DNTC from Ag+ mice
transcribe severely reduced amounts of IL-2 following challenge with
antigenic ligand (Fig. 1B). These cells are also defective
in IL-15 synthesis, but they express equivalent levels of TNF- and
TGF-. However, unlike other models of T cell anergy
(27), DNTC from Ag+ mice do not
express elevated levels of IL-10 (data not shown). The defective
response by DNTC from Ag+ mice to Ag could be
overcome by the addition of either exogenous IL-2 or IL-15 (Fig. 1C). Reconstitution of the proliferative response by IL-15
is likely due to the high expression of IL-2R, a receptor for IL-15
(28), by these cells.
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memory markers
(Fig. 2A). They also parallel
memory T cells in that they do not express the acute activation markers
CD25 and CD69 (Fig. 2A). In addition,
H-2b/d DNTC also expressed the 1B11 epitope, a
recently described marker for effector CD8+ T
cells (29, 30). Further corroborating a memory phenotype,
DNTC from Ag+ mice are capable of synthesizing
high levels of IFN- rapidly after stimulation (Fig. 2B).
By contrast, neither DNTC from Ag- nor naive
CD8+ T cells produced detectable levels of the
cytokine during the same test period (Fig. 2B and data not
shown).
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DNTC from Ag+ mice up-regulate CD25 when
stimulated either by the low- (p2Ca/Kb) or the
high- (p2Ca/Ld) affinity ligands (Ref.
20 ; Fig. 3A). By
contrast, DNTC from Ag- mice up-regulate CD25
only in response to the high-affinity ligand. To test whether this
heightened capacity of Ag-experienced DNTC to up-regulate CD25 makes
them effective competitors for growth factors produced during the
course of a normal immune response, we designed a cell coculture
experiment. Ag-stimulated DNTC, labeled with CFSE (31) to
track cellular proliferation, were incubated for 3 days in the presence
or absence of 2C CD8+ T cells and their
proliferation was compared. As the DNTC and added
CD8+ T cells express the same TCR, they can both
respond to the same Ag. DNTC from Ag-
(H-2b) mice proliferated well to Ag alone (Fig. 3B). By contrast, the majority of DNTC from
Ag+ (H-2b/d) mice have died
after 3 days of stimulation, as evidenced by the loss of
CFSE-labeled cells. The few cells that survived responded very poorly
to Ag stimulation (Fig. 3B). Strikingly, the proliferation
of H-2b/d DNTC was greatly enhanced by the
presence of activated 2C CD8+ T cells or
exogenous IL-2, whereas the proliferation of the
H-2b DNTC was not further augmented by these
additions. This result indicates that H-2b/d DNTC
can compete effectively for growth factors produced by neighboring
activated CD8+ T cells.
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C). Most striking was the
reduction in CFSE-labeled CD8+ T cell number in
wells containing H-2b/d DNTC. As exogenous IL-2
did not reverse this suppressive effect, mechanisms other than limiting
amount of this cytokine were sought to explain this suppressive
effect. Mechanism of DNTC suppression
As CD8+ T cells undergoing
lymphopenia-induced homeostatic expansion displaycytolytic activity
(6, 7, 8) and H-2b/d DNTC also express
the cytotoxic effector marker, 1B11, we sought to determine whether
DNTC from Ag+ mice also possess cytolytic
activity. To investigate this possibility, DNTC from either
H-2b/d or H-2b 2C mice were
purified and incubated with 51Cr-labeled
T2-Kb and T2-Ld APCs loaded
with 1 µM p2Ca peptide. Remarkably, H-2b/d DNTC
exhibited spontaneous cytolytic activity against APCs expressing either
the high-affinity (p2Ca/Ld,
KA = 2 x 106
M-1) or low-affinity
(p2Ca/Kb, KA = 3
x 103 M-1) ligands (Ref.
23 ; Fig. 4A). By
contrast, H-2b DNTC displayed no killer activity
toward p2Ca/Kb-bearing targets and a very weak,
if any, cytolytic activity toward
p2Ca/Ld-expressing ones.
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B). This killing was specific for
the p2Ca peptide, as the addition of the Kb
binding peptide pOVA (16, 24) did not render them
susceptible to lysis. By contrast, neither Ag-activated
H-2b DNTC nor H-2b 2C
CD8+ T cells could kill these targets even when
they were supplemented with the p2Ca peptide. This finding indicates
that H-2b/d DNTC possess a broader range of
target specificity than either H-2b DNTC or 2C
CD8+ T cells. Both Ag-activated DNTC from
H-2b or H-2b/d 2C mice were
effective killers of DBA/2 (H-2d) Con A blasts,
which express the cognate Ag, in the absence of exogenously added p2Ca
peptide (Fig. 4B). As expected, Ag-activated
CD8+ T cells from H-2b 2C
mice were the most efficient killers of DBA/2 targets.
Two major pathways for T cell mediated cytotoxicity exist, one Fas
based and the other perforin based (32). To determine the
relative contribution of Fas/FasL signaling to DNTC killing of
CD8+ T cell targets, we used both Fas-deficient
targets and recombinant Fas-Fc fusion protein (33) to
abrogate Fas/FasL interaction. Interestingly,
H-2b/d DNTC exhibited greatly reduced killing
activity toward Fas-deficient 2C CD8+ targets as
compared with wild-type (Fas+) targets (Fig. 4C). Corroborating a major role of Fas signaling in DNTC
cytotoxicity, the killing of wild-type 2C CD8+
blasts was blocked by the addition of Fas-Fc fusion protein.
Furthermore, the killing of CD8+ T cells was not
dependent on them expressing the 2C TCR (Fig. 4C).
Nontransgenic CD8+ T cells could be killed as
effectively as 2C TCR-expressing cells. Therefore, the
H-2b/d DNTC killing is dependent upon the
expression of Ag (in this case the low-affinity ligand,
p2Ca/Kb) and Fas on the target cell surface.
DNTC require interaction with cognate Ag to drive their peripheral expansion
As DNTC from Ag+ mice share characteristics
with CD8+ T cells stimulated by homeostatic
proliferation, we sought to test whether these cells may be derived
from a similar process. First, we examined whether cognate Ag
stimulation (H-2b/d splenocytes) of DNTC from
Ag- mice induced the expression of the acute
activation markers CD25 and CD69. In agreement with our hypothesis,
DNTC did not up-regulate these acute activation markers at either
16 h (Fig. 5A) or 40
h (data not shown) post-stimulation, whereas CD8+
T cells rapidly expressed these markers. To test the ability of naive
DNTC to undergo expansion in vivo, we injected CFSE-labeled cells into
sublethally irradiated Ag-
(H-2b/b) or Ag+
(H-2b/d) mice and analyzed their proliferation 1
wk after transfer. As a control for homeostatic expansion, we also
transferred CFSE-labeled CD8+ T cells bearing the
same 2C TCR into sublethally irradiated Ag- or
Ag+ hosts. In agreement with others
(6), we found that 2C CD8+ T cells
undergo homeostatic expansion in Ag- mice (Fig. 5B). In stark contrast to CD8+ T
cells, DNTC from Ag- mice fail to expand in
irradiated Ag- hosts (Fig. 5B). This
observation is similar to one recently describing the fact that 2C DNTC
do not proliferate when placed into lymphopenic RAG1-deficient
(H-2b/b) animals (34). Therefore,
low-affinity interactions of self peptides/MHC of the
H-2b/b background are insufficient to drive DNTC
proliferation. Dissimilar to homeostatic proliferation,
CD8+ T cells injected into
Ag+ recipients undergo rapid proliferation,
completely losing their CFSE fluorescence 1 wk after transfusion (Fig. 5B). By contrast, exposure of DNTC to cognate Ag in vivo
results in a rate of proliferation that is higher than
CD8+ T cell homeostatic expansion but lower than
Ag-driven CD8+ T cell growth (Fig. 5B). In accord with their natural memory phenotype,
H-2b/d DNTC undergo a slow peripheral expansion
after transfer into syngeneic irradiated recipient animals.
Consequently, although H-2b/d DNTC do not respond
well to Ld in vitro, these cells are well
equipped for peripheral expansion in vivo. Collectively, these data
imply that DNTC require high avidity interactions to induce a slow
homeostatic-like growth.
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C). In contrast,
CD8+ T cells undergoing homeostasis-induced
proliferation have an increased incidence of memory-like T cells,
expressing higher levels of CD44, IL-2R, and Ly-6C. Contrary to
cells parked in an Ag- host,
H-2b DNTC exposed to cognate Ag in vivo acquire
an activated/memory state bearing high levels of CD44, IL-2R, and
1B11 (Fig. 5C). However, they do not up-regulate the acute
activation markers CD69 and CD25. By contrast, exposure of
CD8+ T cell to cognate Ag in vivo is associated
with the up-regulation of both acute activation markers (Fig. 5C). This less vigorous response by DNTC to cognate Ag may
be responsible for their resistance to clonal deletion in
Ag+ mice. These results suggest that TCR/cognate
Ag interaction is required for the peripheral expansion of
DNTC.
We have found DNTC from Ag+ mice were able to
respond to the low-affinity (p2Ca/Kb) ligand. To
determine whether DNTC from Ag- mice could
undergo this functional change following homeostatic expansion in
Ag+ mice, we injected purified
H-2b DNTC into Ag+ mice.
Seven days later, the injected DNTC were purified from
Ag+ hosts and were stimulated with either the
low- or the high-affinity ligand of the 2C TCR ± exogenous IL-2.
This response was compared with proliferative responses of DNTC, which
were parked in Ag- mice, or by naive DNTC from
Ag- mice (Fig. 6).
As expected, naive DNTC from H-2b 2C TCR
transgenic mice responded well to the high-affinity
(p2Ca/Ld) ligand regardless of whether exogenous
IL-2 is added. However, these cells lacked the capacity to respond to
the low-affinity (p2Ca/Kb) ligand even in the
presence of exogenous IL-2. By contrast, DNTC that had undergone
homeostatic expansion in Ag+ hosts gave a low
proliferative response to both the high- or low-affinity ligands in the
absence of an exogenous source of IL-2. Strikingly, this poor response
toward either ligand was abrogated by the addition of exogenous IL-2
(Fig. 6). Interestingly, DNTC isolated from Ag-
hosts also responded weakly to p2Ca/Kb plus IL-2
even though they had proliferated minimally after transfer.
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). We found that these
CD8+ T cells can now respond weakly to the low
affinity-ligand plus IL-2. The CD8+ T cells
recovered from Ag- mice also mounted a stronger
response toward p2Ca/Ld as compared with naive 2C
CD8+ T cells. By contrast,
CD8+ T cells recovered from
Ag+ mice proliferated poorly toward
p2Ca/Ld, and the addition of IL-2 had no effect
on this response. This observation is consistent with the observation
that CD8+ T cells recovered from
Ag+ mice are functionally anergic and that this
state is irreversible by exogenous IL-2 (19). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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after
TCR stimulation, and ex vivo cytolytic activity. However, DNTC from
Ag+ mice differ from conventional T cells or
those from Ag- mice in that they respond poorly
when challenged with cognate Ag in vitro and fail to synthesize IL-2.
As they possess potent cytolytic activity and express cognate Ag, their
poor proliferation in vitro may be in part due to fratricide. Despite
the potential for self-self killing, the fact that exogenous IL-2 can
overcome this proliferation defect strongly suggests that diminished
cytokine production plays the prominent role in their defective
response. Consequently, these regulatory DNTC are dependent on
stimulation by both self Ag and bystander-produced cytokine for their
proliferation and heightened cytolytic activity. Furthermore, the
inability to synthesize cytokines required for their proliferation
limits their autoimmune potential. The reliance on the Fas-based
pathway of cytotoxicity may focus DNTC killing on activated T cells or
APCs that have up-regulated Fas expression. The aforementioned
properties make DNTC particularly adept at sensing their environment,
directing their killer/suppressor function toward cells mediating
immunity or autoimmunity. Because this slow expansion/differentiation process appears similar to one recently described for homeostasis-induced proliferation of naive CD8+ T cells (5), we sought to determine whether they may share a similar derivation. To verify this hypothesis, naive DNTC were adoptively transferred to an Ag+ host for 1 wk. After this test period, these previously naive T cells had undergone a slow expansion and memory conversion. Interestingly, these DNTC had not only acquired most of the markers present on DNTC from Ag+ mice, but they had also adopted similar functional attributes. Although it is still not clear whether DNTC that have developed in the presence of Ag (i.e., H-2b/d DNTC) are completely equivalent to naive DNTC that have recently been exposed to Ag for a short period of time (i.e., H-2b DNTC placed into a H-2b/d animal for a period of 1 wk), we do find the similarities, in particular the functional attributes, between these two cell types to be impressive. The fact that either cell type can proliferate robustly to either the low- or high-affinity ligand when supplied with exogenous IL-2 is striking. In addition, Ag-experienced DNTC, regardless of origin, share the expression of a wide array of markers. Collectively, our results suggest that a process resembling a mechanism for maintaining peripheral CD8+ T cell numbers may be used to ensure self tolerance. These observations provide a rationale and basis for the persistence of autoreactive T cells among the repertoire of healthy individuals.
Recently, it has been demonstrated that DNTC clones from TCR transgenic mice possess suppressor-like activity and mediate Ag-specific suppression (35). Although suppression by DNTC clones also involved Fas/FasL interaction, their proposed mechanism of killing differs. According to their model, DNTC clones obtain Ag from APCs through TCR-mediated endocytosis, and they re-express these newly acquired molecules on their cell surface. Subsequently, cognate Ag-specific CD8+ T cells become susceptible to cytotoxicity by their recognition of cognate Ag on the cell surface of DNTC clone, initiating Fas/FasL signaling and programmed cell death. However, our data does not fit with such a model, as H-2b/d DNTC can kill either 2C TCR-expressing or nontransgenic CD8+ T cells, which express a wide array of TCR, with equivalent efficiency. Therefore, the killing of CD8+ T cells by DNTC is not dependent on recognition of a cognate Ag on DNTC by the CD8+ T cell. In our studies, killing is dependent on recognition of the Ag on the target cell by the TCR of the DNTC. Exposure of DNTC to self Ag in vivo results in a lowering of the triggering threshold (20). This enables DNTC to respond to a greater variety of self Ags and hence, to regulate the response of a larger repertoire of T cells. Therefore, DNTC may play an important role in maintaining peripheral T cell tolerance and down-regulating immune responses by killing activated T cells, particularly those reactive against self Ags. The dominant role of the Fas/FasL in DNTC mediated suppression suggests that the inability of such regulatory T cells to function may contribute to the autoimmunity observed in animals that are deficient in either Fas (lpr/lpr) or FasL (gld/gld) expression (36, 37).
DNTC are relatively rare in healthy mice, comprising 3–5% of the
peripheral T cell pool. The heightened incidence of DNTC in
lymphoproliferative, graft-vs-host, and autoimmune disease suggests
either a pathogenic or immunoregulatory role for this subset of T cells
(38). The origin of DNTC in TCR transgenic mice has been a
subject of considerable interest. Although DNTC in TCR transgenic mice
have been suspected to arise from the forced expression of the
transgene in 
precursors (11), it is possible that
they instead represent a distinct lineage of cells. Our analyses
of spleens from normal mice revealed that although both and
DNTC possess similar cell surface markers as regulatory DNTC
expressing the 2C TCR, only activated DNTC from normal mice are
effective killers of syngeneic CD8+ T cell blasts
(data not shown). Interestingly, NK T cell
(NK1.1+ DNTC) killing was also biased toward the
Fas/FasL-based pathway. In contrast to mainstream T cells,
CD4+ and
CD4-CD8- NK T cell
subsets exhibit a restricted TCR diversity and are largely restricted
by the invariant CD1 molecule (39). Recently, the mouse
CD1-restricted repertoire has been described to be dominated by a few
autoreactive TCR families (40). The fact that these cells
possess semivariant, autoreactive TCRs and a natural memory
phenotype suggests that they may be derived from a self Ag-driven
selection and peripheral expansion (39, 40). Additionally,
CD1 has been implied to play a critical role in the deletion of
activated CD8+ T cells (41). Based
on the similarities between regulatory DNTC from 2C mice and
CD4-CD8-NK1.1+TCR+
cells in normal mice, it is conceivable that the forced expression of
the transgenic TCR in immature T cells may have induced their
differentiation along the NKT cell lineage. In summary, we have shown
that self Ags mediate the peripheral expansion and conversion of
DNTC from TCR transgenic mice into potent immunoregulatory cells. These
self-reactive regulatory cells are uniquely poised to suppress immune
responses and prevent autoimmunity. The existence of DNTC in normal
mice with a similar cell surface phenotype and cytolytic function
suggests that a similar process may operate in normal mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, University of British Columbia, Room 300 Westbrook Building, 6174 University Boulevard, Vancouver, Canada, V6T 1Z3. E-mail address: teh{at}interchange.ubc.ca
3 Abbreviations used in this paper: DNTC, double-negative (CD4-CD8-+) T cell; LN, lymph node; ccDNA, competitor cDNA.
Received for publication June 19, 2001. Accepted for publication September 24, 2001.
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T cell receptor can replace the receptor in the development of lineage cells. Immunity 5:343.[Medline]
T cells that contains autoreactive cells. J. Exp. Med. 174:1001. T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4-CD8- T cells in antigen-expressing mice. J. Immunol. 163:1222.8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172:1091.-chain of the interleukin-2 receptor. Science 264:965.+) T cells which promote tolerance induction and regulate autoimmunity. Immunol. Rev. 149:217.[Medline]
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