By
§
From the * Department of Immunology, College of Medicine, Jagiellonian University, Krakow, Poland; Section of Allergy and Clinical Immunology, Dept. of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut 06520-8013; § Department of Biology and Section of
Immunobiology, Kline Biology Tower, Yale University, New Haven, Connecticut 06520; and
Imperial
Cancer Research Foundation, London, England WZCA 3PX
Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are
clearly subject to multifaceted regulation. There is abundant evidence that downregulation of
CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation,
we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using T cell-deficient (TCR
/
) mice, are presented here.
The results clearly show that TCR
/
mice cannot mount CS, implicating
T cells as the
critical CS-effector cells. However, TCR
/
mice can, after high dose tolerance, downregulate
+/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then
adoptive cell transfers in vivo, the active downregulatory cells in tolerized
/
mice are
shown to include
TCR+ cells that also can downregulate interferon-
production by the targeted CS-effector cells in vitro. Downregulation by
cells showed specificity for hapten,
but was not restricted by the MHC. Together, these findings establish that
T cells cannot
fulfill CS-effector functions performed by
T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by
T cells. Comparisons are likewise considered with downregulation by
T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic
autoimmunity.
Cutaneous contact sensitivity (CS)1 responses to contact
sensitizing hapten Ag such as picryl chloride (PCl) and
oxazolone (OX) are classical manifestations of T cell-mediated immunity in vivo. In previously sensitized hosts, CS
and the related delayed-type hypersensitivity (DTH) reaction, are manifest as macroscopically measurable inflammation (skin swelling) that peaks at 24-48 h after topical cutaneous Ag challenge (1).
It appears that early after challenge with hapten, local tissue mast cells (2, 3) and blood platelets (4) are induced to
release the vasoactive amine serotonin (2) that facilitates local extravasation and subsequent migration and activation
of Ag-specific CS-effector lymphocytes. Available data
strongly suggest, but do not prove, that the responding lateacting lymphocytes are exclusively MHC class II-restricted,
By contrast to the conventional CS-effector response,
which is induced by immunization via skin painting with
reactive hapten Ag, mice respond to high intravenous doses
of soluble hapten Ag by developing tolerance. This high
dose Ag tolerance is of particular significance with regard
to the capacity of individuals that become tolerant toward
contact hapten allergens, and may also underlie aspects of
tolerance to self Ag. Numerous studies have demonstrated that high dose tolerance can be associated with the development of regulatory T cells that limit the response of CSeffector T cells (12). In this regard, it is unclear whether
or not such CS-downregulatory T cells are exclusively An assay commonly used to demonstrate high dose downregulation of CS, is the mixing together in vitro of spleen
cells from mice tolerized intravenously with high doses of
Ag, together with CS-effector cells from contact sensitized
mice. Thereafter, the mixed regulatory and CS-effector cells
are adoptively transferred to naive recipients, and a subsequent measurement is made of the transferred CS-effector
response in the recipients. The studies reported in this
manuscript use this mixing assay and other established assays, along with use of TCR-deficient Mice.
5-7-wk-old 129/J (H2b), BDF1 (H2b × d), C57Bl/6
(H2b), BALB/c (H2d), and CBA/J (H2k) mice were obtained
from Jackson Labs (Bar Harbor, ME). TCR Reagents.
Picryl chloride TNP chloride (PCl) obtained from
Chemica Alta (Edmonton, Alberta, Canada) was recrystallized
from methanol/H2O, and protected from light and humidity.
Trinitro-benzene sulfonic acid (TNBSA) (Eastman Fine Chemicals, Rochester, NY); oxazolone (4-ethoxymethylene-2-phenyloxazolone) (OX) (British Drug Houses via Gallard-Schlesinger
Inds. Inc., Carle Place, NY); and anti-hamster IgG antibodycoated magnetic beads (1 µm iron magnetic particles; Advanced
Magnetics, Inc., Cambridge, MA) were obtained from the manufacturers. Anti-TCR Active Immunization.
Mice were actively sensitized by topical
application of 0.15 ml of 5% PCl or 3% OX in 1:3 acetone-ethanol mixture, to the shaved abdomen, chest, and hind feet on day
0. 4 d later, zero time ear thickness was measured with an engineer's micrometer (Mitutoyo Mfg. Co., Tokyo, Japan) before local ear skin challenge via topical application to both sides of both
ears of one drop of 0.8% PCl or OX solution in olive oil (for 129/J,
BALB/c, TCR Induction of High Ag Dose Tolerance.
For induction of TNP-
specific high dose tolerance, mice received two intravenous injections of 3 mg TNBSA (1% TNBSA in distilled H20, readjusted
to pH 7.2 with 1 M NaOH) in 0.3 ml on days 0 and 3. On day 7, spleens from these TNBSA-injected mice were harvested as a
source of putative regulatory cells. Alternatively, mice were tolerized by intravenous injection of TNP-conjugated syngeneic spleen cells.
TCR+, CS-effector T cells (7, 8), which customarily
make Th1-type effector cytokines, such as IFN-
(9).
T cells, or whether they may include
T cells.
/
mice, to assess
the cells responsible for CS elicitation and for CS downregulation, respectively. The studies show that
T cells cannot substitute for
T cells as effectors of CS elicitation, but
that
T cells can medite downregulation of both
CS effectors in vivo and IFN-
production by these CS effectors
in vitro. The studies are particularly pertinent given the reported capacity of
T cells to negatively regulate
T
cell-driven responses in allergic (18, 19) and other immune
responses.
/
and TCR
+/
mice (20) with different but defined MHC backgrounds that
included H2b, H2d, and H2b+d, were supplied locally. All mice
were maintained in microisolator cages, and changed in a laminar
flow hood. MHC haplotypes were determined by FACS® with antiMHC class Ib (PE-conjugated), and class Id (FITC-conjugated)
mAb (PharMingen, San Diego, CA).
mAb (clone UC7-135D5, hamster IgG), was obtained from Dr. J. Bluestone (University of Chicago, Chicago, IL; 23). Low-tox rabbit complement C was obtained from
Pel-Freeze Biologicals (Brown Deer, WI).
+/
and
/
), or 1% PCl to less reactive BDF1
or C57Bl/6. The subsequent optimal increase in ear thickness
was measured at 24 h, and occasionally 48 h, after challenge as reported (1), and expressed in units of 0.01 mm ± SE. In all experiments, a group of nonimmune control animals was also challenged on the ears with 0.8% PCl or OX, and the resulting
background increase in ear thickness (~2 U at 24 h) was subtracted from responses of experimental groups to yield net ear
swelling responses, shown in the figures.
Adoptive Cell Transfer of CS Responses and Cell Mixing Assay. Donors of CS-immune effector cells were contact sensitized with 5% PCl or 3% OX on days 0 and 3. Immune lymph node and spleen cells were harvested on day 7 and 7 × 107 were injected intravenously into normal syngeneic recipients. Immediately after transfer, recipients were challenged on the ears with 0.8% PCl or OX in olive oil. The increase in ear swelling was determined 24 h, and sometimes 48 h later. It was compared to actively sensitized positive controls and to negative controls that were simply challenged on the ears with 0.8% PCl or OX.
For the cell mixing assay, 5-7 × 107 CS-effector immune lymph node and spleen cells from PCl or OX contact sensitizedTransfer into Tolerized Mice to Confirm Active Tolerance and Agspecificity.
To test tolerance and Ag specificity of downregulatory cells in another system, /
mice were tolerized with a
high dose of TNBSA or with OX-MRBC intravenously on days
0 and 3 before serving as cell transfer recipients. Then on day 7, nontolerized control or TNP- or OX- tolerized
/
mice received 7 × 107 CS-effector cells from normal TCR
+/+ mice
(BALB/c) that were Ag homologous, or were of unrelated Ag specificity (TNP versus OX).
/
recipient mice were then
challenged immediately on the ears with homologous hapten
(TNP or OX), and ear tested for 24 h ear swelling.
Immuno-magnetic Bead Cell Fractionation.
Spleen cells (1.5 × 108) from four TNBSA-tolerized +/+ 129/J, or
/
donor
mice were incubated in 20 ml UC7 hamster anti-TCR
mAb (12 µg/ml) hybridoma supernatant for 60 min on ice. After mAb coating, cells were washed three times with PBS + 2% FCS, and resuspended in 40 ml PBS + 2% FCS containing goat anti-hamster Ig-coated paramagnetic beads at 5-10 beads per target cell.
The cells were then incubated in a flat vertical 50-ml flask for 30 min on ice, after which a magnet (Advanced Magnetics Inc.,
Cambridge, MA) was applied to one side of the flask. 10 min
later, magnetic bead nonadherent cells were recovered, and then the
magnetic bead adherent cells were recovered. Nonseparated regulatory cells were used in experiments shown in Figs. 3, 5, and 6 b.
Recoveries of
Mixing of CS-effector Cells and Magnetic Bead-enriched Regulatory
Cells.
Immune CS-effector cells (7 × 107) were incubated with
5 × 107 unseparated regulatory T cells, with an equivalent number of separated magnetic bead positive cells, or with an
equivalent number of magnetic bead nonadherent
cells (i.e.,
TCR+-remaining cells) originating from either tolerized 129/J
+/+ mice or TCR
/
mice for 30 min at 37°C. The washed
effector and regulatory cell mixture was then injected intraperitoneally into naive 129/J recipients that were tested for CS responses by challenging ears with 0.8% PCl or OX in olive oil the
next day. Recipients were then measured for ear swelling 24 h after Ag challenge to the ears.
In Vitro Culture of CS-effector Cells and Hapten-conjugated APC
for Elaboration of IFN-, and Regulation by Tolerized Cells.
A single cell suspension of just CS-effector lymph node cells was obtained aseptically from PCl contact sensitized mice. Suspensions of regulatory cells were from spleen cells of mice that were injected with TNBSA or OX-MRBC intravenously on days 0 and 3, or from control nonimmune mice. In some experiments, spleen
cells from TNBSA-tolerized mice, or nonimmune controls, were
incubated with 1 µg biotinylated anti-
TCR (GL3; PharMingen) per 106 cells at 2 × 107 cells/ml for 20 min on ice. Then, 10 µl
of streptavidin-conjugated magnetic microbeads (Miltenyi, Sunnyvale, CA and BioTek Instrs. Inc., Sunnyvale, CA) per 107 cells
were added and incubated for 15 min on ice. After washing, this
cell and bead suspension was applied to a MiniMACS magnetic separation column (Miltenyi). After recovering of nonadherent
-depleted cells and washing the column,
-enriched adherent cells were eluted. Equivalent numbers of
-enriched, or
depleted cells were added in vitro as regulatory cells, to the CSeffector cells (1:1; 2 × 105/well), and then were added together
for 72 h to mitomycin C-treated TNP- or OX-conjugated normal spleen cells as APC (2 × 105) in 0.2 ml of RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 25 mM Hepes, 5 × 10
5 M/2-mercaptoethanol, and
10% fetal calf serum. At 48 h, culture supernatants were collected
for IFN-
quantitation.
ELISA Quantitation of IFN- in Culture Supernatants.
Quantitative sandwich ELISA for IFN-
was performed with two different mAb specific for mouse IFN-
(clones R4-6A2 and
XMG1.2; Pharmingen). 1 µg/ml of capture mAb to IFN-
in
0.1 M Na2CO3, pH 8.3, was briefly coated onto 96-well microtrays
(Corning Glass Incorporated, Corning, NY) overnight at 4°C. After blocking with 3% dry milk in PBS, samples and standard recombinant mouse IFN-
(Genzyme Corp., Cambridge, MA)
were applied to the wells and incubated overnight at 4°C. Then,
0.5 µg/ml of biotinylated separate mAb to IFN-
was applied for
45 min at 25°C, followed by incubation with 1:3,000 dilution of
horseradish peroxidase-conjugated streptavidin (Vector Labs.,
Inc., Burlingame, CA), for 30 min at 25°C. TMB (tetramethylbenzidine) microwell peroxidase substrate, and TMB one component stop solution (Kirkegaard & Perry Laboratories, Inc.,
Gaithersburg, MD) were used for reaction development and were
read at OD 405.
Statistics. Double-tailed Student's t test was used to assess the significance of differences between groups, with P <0.05 taken as a minimum level of significance.
CS responses
in contact sensitized TCR/
mice and control 129/J mice
were compared by sensitizing (via abdominal skin painting) with concentrated PCl (5%), followed 4 d later by topical
challenge with dilute PCl (0.8%) applied to the ears, and
subsequent measurement of ear swelling 24 h later. It was
found that
T cell-bearing 129/J mice developed a classical 24 h ear swelling response (Fig. 1, group A), whereas
TCR
/
mice did not (Fig 1, group B).
When /
mice were used as recipients of
+ cells
from MHC-matched, immunized
T cell-sufficient BDF1
mice, they could mount CS upon ear challenge with hapten (Fig. 1, group D), similar to normal mice (Fig. 1, group
C). This demonstrated that the vascular and cellular environment for eliciting CS was normal in TCR
/
knockout mice. The finding that the 24 h component of CS cannot be elicited in actively sensitized TCR
/
mice is
consistent with other studies indicating that the active CSeffector cell type is TCR
+ (7, 8, 24).
Because TCR/
mice
do not mount CS responses, it is impossible to determine
directly whether high dose Ag tolerization of such mice induces downregulatory cells. Instead, CS-effector cells (7 × 107) from contact-sensitized 129/J mice were mixed and
incubated for 30 min at 37°C with 5 × 107 spleen cells
from
/
mice, that were harvested on d 7 after two intravenous injections of tolerogenic TNBSA. Analogous
TNBSA-tolerized cells were derived from TNBSA-treated
MHC haplotype-matched TCR
+/
mice, or TCR
+/+
(129/J) mice. The cells were then transferred back to haplotype-matched recipient mice that were challenged with
hapten.
Fig. 2 shows that upon transfer back to recipient mice,
CS-effector cells elicit a 24 h response that can be significantly reduced by preincubation of the effector cells with
cells from tolerized TCR+/+ mice (Fig. 2, group B),
TCR
+/
mice (Fig. 2, group C), or TCR
/
mice (Fig.
2, group D). Essentially the same results were obtained when effector cells from sensitized BDF1 mice were mixed
with cells from either TNBSA-tolerized BDF1 mice or
TNBSA-tolerized TCR
/
mice (Fig. 2, groups E-G).
A prediction that follows from the data shown in Fig. 2
(groups A-G) is that TCR/
mice will not support the
actions of adoptively transferred TCR
+/+ CS-effector
cells if the recipient
/
mice are subject to high dose Ag
tolerance beforehand. The data in Fig. 2, groups H and I
confirm this prediction. This result is important in that it
rules out that the detection of TCR
/
downregulatory
cells in adoptive transfer experiments (Fig. 2, groups D and G)
was artefactual, due to manipulation of the cells in vitro
and their subsequent transfer. Furthermore, induction of
high dose tolerance by TNBSA in TCR
/
mice was reproducibly demonstrated using transfers from individual TCR
mice (data not shown), indicating the generality
of the findings.
To determine the phenotype of CS-regulatory cells induced by high Ag dose tolerance, splenocytes from TNBSA-tolerized mice were fractionated by use
of anti-TCR mAb-coated magnetic beads.
+,
, or
unfractionated tolerized cells were then mixed for 30 min at 37°C with lymph node and spleen CS-effector cells (7 × 107) from contact immunized, haplotype-matched mice,
and the mixture transferred to recipients that were subsequently assayed for CS responses by ear challenge. In these
experiments, anti-
bead adherent cells ranged from 55 to
95%
TCR+ when reanalyzed by FACS® (see Materials
and Methods). Thus, we estimate that
106
cells were
adoptively transferred into each recipient when unseparated TCR
/
cells were used, and about
7 × 105
cells were transferred when magnetic bead
-enriched
cells were used.
The data in experiments 1 and 2 of Fig. 3 show that
while the cells from TNBSA-treated +/+ 129/J mice that
are responsible for downregulating CS effectors are exclusively TCR
(group D), the most potent regulatory cell
fraction from tolerized TCR
/
mice scored as TCR
+
(Fig. 3, group F). Similar results were obtained in experiment three, in which cells from H2d TCR
/
mice were
compared with cells from H2d BALB/c mice for their response to high dose Ag tolerance. As before, when measuring the capacity of tolerized cells to downregulate a CSeffector response to PCl, the TCR
+ fraction from
TCR
/
mice, but not from
+/+ BALB/c mice, was
again active (experiment 3, group C). Conversely, the active fraction from normal
/
mice was TCR
+ (group
D), and the TCR
fraction from TCR
/
mice
(group E) contained negligible residual activity.
The discovery that T cells in TCR
/
mice
can mediate downregulation of CS effector cells prompted
an investigation of Ag specificity, since
T cells and
cells appear to have a different set of Ag specificities. To
test this, cells from TCR
/
mice tolerized with high
doses of OX or TNP, were mixed with CS-effector cells
from TCR
+/+ mice sensitized with PCl or OX, and
compared in their capacities to downregulate the 24 h CS
response after transfer back to recipient mice. Data presented in Fig. 4 a show no capacity of cells from OX-tolerized TCR
/
mice to influence the CS response to TNP
(group B), whereas TNP responses were inhibited significantly after mixing effector cells with cells from TNP-tolerized TCR
/
mice (Fig. 4 a, group C). Data in Fig. 4 a,
groups D-F demonstrate that reciprocal Ag specificity was
likewise apparent (i.e., cells from OX-tolerized TCR
/
mice downregulated effector cells from OX-sensitized mice,
but not
+/+ TNP-immune cells) (group E versus F).
To confirm the above findings we tested Ag-specificity
in tolerized /
mice by attempting to transfer
+/+
CS-effector cells into these recipients.
/
mice were tolerized with a high dose of TNBSA or OX-MRBC intravenously before cell transfer. Then, on day 7, control (nontolerized) or TNP or OX-tolerized
/
mice received 7 × 107 TNP or OX immune cells from normal
+/+ (BALB/c)
mice. The data presented in Fig. 4 b show there was no
ability to adoptively transfer immunity into TNP tolerized
/
mice with TNP-specific CS-effector cells (group B),
while TNP-immune CS-effector cells transferred normal
CS responses into OX-tolerized
/
recipients (group C).
Similar data were obtained when OX-specific immune
cells were transferred (group D versus F). Thus, total inhibition of adoptive cell transfer was obtained when OXspecific immune CS-effector cells were transferred to OXtolerized
/
mice (Fig. 4 b, group E), whereas OX CS
was successfully transferred to TNP-tolerized
/
mice
(Fig. 4 b, group F). Thus, this system that did not involve cell harvest, cell mixing, nor cell transfer, confirmed the
presence of active Ag-specific downregulation in high
dose-tolerized
/
mice.
To determine possible MHC restriction of downregulatory T cells, magnetic bead-enriched
TCR+ cells from
TNBSA-tolerized
/
mice were incubated with 7 × 107 PCl-immune CS-effector cells from MHC-compatible
(129/J, H2b), or -incompatible (CBA/J, H2k) mice, for 30 min at 37°C, followed by subsequent transfer to recipients
syngeneic with the CS-effector cells. The recipient mice
were challenged immediately (before any significant host response to the allogeneic
graft could develop), and ear
swelling responses determined. The results in Fig. 5 show
that
+ cells from TNBSA-tolerized TCR
/
mice
downregulated CS-effector responses of allogeneic CBA/J H2k CS-effector cells (group D), as effectively as the regulation of H2-matched CS-effector cells (group B).
To establish the phenotype of T cells capable of
inhibiting CS-effector responses, spleen cells from TNBSA-tolerized
/
mice were treated with anti-CD4 or
anti-CD8 mAb or PBS, and then rabbit complement 1:75.
The three resultant populations of TCR
/
cells were
then individually incubated with 129/J immune CS-effector cells for 30 min at 37°C, and each mixture of cells was
subsequently assayed for CS-effector function after transfer
to naive recipients. The results in Fig. 6 a demonstrate once
more that TNBSA-treated TCR
/
mice are a reliable
source of downregulation of CS-effectors (group B), and
that this activity is unaffected by treatment with either antiCD4 (group C), or anti-CD8 (group D). Results are consistent with the active regulators being double negative
(DN)
T cells.
DN T cells have been reported in the lymphoid system of essentially all vertebrates examined, but they occur
in mice in very low numbers relative to
T cells. Therefore, to assess the potency of DN
T cells to downregulate CS, a dose-response experiment was performed. 10fold dilutions (2.5 × 105, 2.5 × 104, or 2.5 × 103) of 60%
TCR+ cells (purified from TNBSA-tolerized
/
mice by use of magnetic beads) were incubated, each with
7 × 107 PCl-immune CS-effector 129/J cells for 30 min at
37°C, and then were harvested, washed, and transferred.
Recipient ear swelling results (Fig. 6 b) showed a strong
dose response of the assay to the inocula (groups B-D).
Even the dose of 2.5 × 103
T cells had activity in the
assay (group D), albeit weaker than the other doses. The
capacity of very small numbers of downregulatory
T
cells to score in this bio-assay is discussed below.
Since IFN- is a central cytokine in Th-1-mediated responses, such as DTH and CS, we determined
whether IFN-
production by CS-effector TNP-immune
cells was affected by regulatory cells induced by high dose
intravenous TNBSA or OX tolerogenesis. Using two different anti-IFN-
antibodies in an ELISA assay, it was first
established that 2 × 105 lymph node cells from contact immunized mice, stimulated in vitro by TNP-conjugated
APC, secreted >3 ng/ml IFN-
over a 48 h period. Compared to production of IFN-
by CS-effector cells mixed
with nonimmune spleen cells (Fig. 7 a, groups A and D),
IFN-
production by CS-effector cells and TNP-APC was
substantially inhibited by mixing with spleen cells from intravenous TNBSA tolerized normal
+/+ mice (Fig. 7 a,
group B) and also with spleen cells from intravenous TNBSA
tolerized
/
mice (Fig. 7 a, group E). Also, as controls,
spleen cells from nonimmune and nontolerized animals incubated alone or in the presence of TNP or OX-APC did
not secrete detectable levels (0.1 ng) of IFN-
(data not
shown).
To determine the phenotype of regulatory cells responsible for the in vitro suppression of IFN- production,
TCR+ or
TCR
cells in the spleens were separated, using anti-
antibody coated magnetic beads that were used
to separate spleen cells from nontolerized, TNBSA-tolerized, or OX-tolerized TCR
+/+ or TCR
/
mice, and
then equivalent numbers of each cell subpopulation were
added to TNP-immune CS-effector cells in vitro, and cultured together with TNP-hapten-conjugated APC. There
was a striking suppression of IFN-
production by
+
cells from TNBSA-tolerized
/
mice (Fig. 7 b, group E,
left) that did not occur with
+ cells from TNBSA-tolerized
+/+ mice (Fig. 7 b, group B, left). In contrast, the inhibitory activity of TNBSA-tolerized
+/+ mice was shown
to be due to
cells (Fig. 7 b, group B, right), and thus
was probably due to
T cells. In contrast to the downregulatory activity of
+ cells from TNBSA-tolerized
/
mice, the
cells of these mice were totally without inhibitory activity (Fig. 7 b, group E, right), as were OX-tolerized
+ and
cells (Fig. 7 b, group C and F, left),
again varifying Ag-specificity of
+ cells mediating tolerance.
These in vitro findings were consistent with the in vivo
findings. Thus, we concluded that suppressive cells in Agtolerized /
mice were potent Ag-specific
downregulatory T cells, both in vivo with contact sensitivity, and in
vitro with IFN-
production, while T cells that inhibited
CS in vivo and IFN-
in vitro that were obtained from
normal
+/+ mice, were not
T cells, and probably were
T cells. The failure of most cell mixtures tested in Fig. 7 b
to reduce IFN-
production demonstrated that selected reductions in IFN-
production were not readily attributable
to nonspecific effects of extra cells in the assay, such as
binding of IFN-
to IFN-
receptors, or to degradation of
secreted IFN-
.
T cell-deficient mice do not elicit the classical 24 h
component of CS after active contact sensitization and subsequent skin challenge. This result extends previous findings implicating
T cells as principal effectors in CS responses (7, 8, 25, 26): i.e.,
T cells and
T cells are not
redundant in their functional capacity as effector cells in
CS. By contrast, TCR
/
mice can respond to high dose
Ag tolerance by the induction of cells that can downregulate CS-effector activity from
+/+ immunized donors.
This was shown in two ways: splenocytes from tolerized
TCR
/
mice downregulate CS-effector cells with which
they are mixed in vitro, before cotransfer and elicitation of
CS, and high dose Ag-treated tolerized TCR
/
mice inhibit the transfer of CS-effectors that function normally in
untreated TCR
/
mice.
The experiments presented here demonstrate that cell-
enriched fractions from TCR
/
mice are better mediators of high dose Ag tolerance than are
cell-depleted
fractions, suggesting that
T cells are the active regulatory
cells, as opposed to TCR
+
cells that are a potential feature of TCR
/
mice (27). The lack of conventional
MHC restriction shown by the TCR
/
regulatory cells,
and their CD4
CD8
phenotype are both consistent with
most assays of
function (29). The apparent hapten specificity of the regulatory
cells demonstrated herein for
the first time may be considered consistent with the hypothesis that the potential diversity (30) of the
TCR
is greater than that of any other Ag receptor (31). Thus, overall, downregulation can be mediated by Ag-specific
T cells, and need not be solely mediated by specific
T cells. However, in this study we have provided no evidence that the
cells directly recognized either PCl/TNP
or OX, nor whether the
cells recognized either conventional APCs, or the effector T cells with which they were
mixed, or some other cells. Conceivably all of these cells may have been haptenated.
Commonly, it has been argued that TCR/
mice are
deficient in immune effector responses because
cells are
uniquely capable of specifically responding to the challenging antigen. However, the apparent capacity of
cells in
TCR
/
mice to distinguish between CS-effector cells
that are responsive to different hapten Ag raises questions
about the completeness and/or validity of this explanation.
Possibly, potential
effector cells responsive to challenge
by specific Ag may be unable to mount strong immune effector responses because they cannot interact efficiently
with the professional antigen presenting cell system. This
may be for physiologic reasons; e.g.,
cells may occur in
an inappropriate anatomical location, in insufficient numbers, or produce inappropriate effector molecules. However, equally attractive is the idea that
cells, like B cells,
only rarely recognize molecular complexes of antigenic
peptides and MHC. In such a case,
cells would not
"learn" that the body was infected from APCs and could
not be expected to mount a conventional immune response of the kind that we associate with APC-
T cell
interactions (31).
Provocatively, the data presented here bear striking similarities with the role of cells reported in another system,
namely the IgE allergic responsiveness of mice to protein
antigen aerosols (18, 19). Thus
T cells reduced ovalbumin-specific IgE responses in rats and mice that were tolerized with ovalbumin administered via repetitive respiratory
aerosol administration (18, 19). Not only were
T cells
shown to be active in both cases, but strikingly low numbers of
T cells per animal (~2 × 103) were effective, as
also was the case here (Fig. 6 b). Likewise, systemic oral tolerance to ovalbumin was suggested to be due to
T cells
(32). Recently it was also shown that mice deficient in
T cells, and responding to infection with a natural intestinal pathogen, Eimeria, had exaggerated gut immunopathology
attributable to unrestrained
T cell function, or the effects thereof, suggesting a downregulatory function of
T cells (33). In other studies,
T cells from T. cruzi infected mice were shown to be suppressive in vivo and in
vitro, correlating with autoimmunity (34). Finally, hepatic
T cells could adoptively transfer tolerance to allogeneic skin grafts, and also reduced lymphokine production (35).
T cells in tumor bearing mice were reported to decrease
anti-tumor cytotoxic T cell activity (36). Thus, the data
presented here, in the classical CS system, provide a clear
demonstration of a fundamental functional phenotype of
cells that is operative in numerous systems, including allergy to aerosolized antigen, oral tolerance, autoimmunity,
transplantation, tumor immunity, and pathogenesis of infectious diseases. As such, the downregulation of
T cells
by the effects of
T cells is emerging as a major and probably general functional capacity.
The downregulatory effect of T cells that we describe
may in part be effected by a direct or indirect inhibition of
IFN
production by the CS-effector cells, as our results
suggest. The mechanism for this inhibition has yet to be resolved. An obvious possibility is the production by
T cells
of cytokines that inhibit T cell production of IFN-
, such
as TGF
, IL-4, or IL-10. The production by
T cells of
IL-4 would be consistent with the capacity of
T cells
from TCR
/
mice to support copious production of antibodies (22). Thus, regulation of CS by
T cells may be
another instance of Th2 regulation or diversion of Th1
cells (37). Alternatively,
T cells may regulate CS-effector
T cells by their being eliminated via injury, apoptosis, or cytotoxic lysis. There are many reports that
T cells
possess cytotoxic activity (38, 39). However, it is noteworthy that we have recent evidence that in vitro tolerance for
IFN-
production by splenocytes of TNBSA-tolerized
mice can be reversed by IL-12 (Ushio, H., and P.W. Askenase, unpublished results), suggesting that cytotoxicity may
not be the mechanism for
T cell-mediated TNBSA tolerance.
In summary, DTH and CS are examples of important in
vivo Th1 responses that can play a crucial role in defense
against diverse microbial pathogens and tumor cells, and as
effectors of some allergic and autoimmune diseases. This
may be the reason for such tight regulation of these responses. It is possible that changes in immunoregulation
could be a cause for decreased immune resistance in serious
infections, or in cancers, or perhaps for the abnormally increased immune responses that occur in allergies and autoimmunity. The findings of the current study clearly establish that T cells can function as a newly recognized
component of immune downregulation, and may be able
to do so in an Ag-specific fashion, the underlying mechanism of which needs now to be defined.
Address correspondence to Philip W. Askenase, Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedal Street, New Haven, CT 06520-8013.
Received for publication 3 May 1996
The authors are especially grateful to Scott Roberts (University of Connecticut, Storrs, CT), Topher Dudley (National Institutes of Health), and Adrian Smith (Yale University, New Haven, CT) for review of the manuscript, and to Marilyn Avallone for her excellent secretarial skills.1. |
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