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Department of Immunology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß+, MHC restricted, and hapten specific. The T
cells proliferated in response to APC sensitized in vivo, but not to
APC coupled in vitro with FITC. Cell lines from unirradiated mice were
Th1 like, producing large amounts of IFN-
, but little IL-4 or IL-10,
whereas cloned Ts cells from UV-irradiated mice produced IL-10, but no
IL-4 or IFN-. Ts cells blocked APC functions and IL-12 production in
vitro. Injection of 5 x 104 cloned Ts cells into
untreated recipients suppressed the induction of CHS. These results
suggest that UV radiation can induce a distinct T regulatory type
1-like Ts population that may block the activation of Th1 cell-mediated
immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the candidate mediators of suppression are cutaneous APC, such as Langerhans cells and dermal dendritic cells, which are thought to play a central role in immunosurveillance by inducing T cell immune responses to tumor Ags and other Ags in the skin (4, 5). Following the application of a contact-sensitizing agent to skin, Ag-bearing Langerhans cells migrate to the draining lymph nodes, where they activate T lymphocytes (6). Lymph node cells from unirradiated, FITC-sensitized mice transferred to unimmunized, syngeneic recipients induce an Ag-specific contact hypersensitivity (CHS)5 response (7). UV radiation depletes the number of Langerhans cells in skin and alters their Ag-presenting function, resulting in the generation of suppressor T (Ts) cells (8, 9). Cells transferred from the lymph nodes of UV-irradiated, FITC-sensitized mice fail to induce a CHS response to the hapten and result in the production of Ts cells in the spleens of these recipient animals (10, 11).
The pathways by which UV radiation alters APC functions and induces Ts
cells are incompletely understood. UV-irradiated APC differentially
activate Th subsets, failing to stimulate Th1 cells, but activating Th2
cells (12, 13). Because Th1 cells, which secrete IFN-, can mediate
CHS responses to hapten, reduction of their function would explain the
impairment in T cell immunity (12, 14). Th2 cells produce IL-4, IL-5,
and IL-10, and suppress the activation of Th1 cells, and are thought to
be Ts cells (15, 16, 17, 18).
Recent studies revealed that, although APC in lymph nodes of UV-irradiated mice were functionally deficient, transferable suppression was mediated by a small number of CD4+ T lymphocytes that copurify with donor FITC-bearing APC (11). However, whether the donor T cells were Ts precursors or induced Ts cells in the recipients was unknown, as was the mechanism of suppression.
To address these questions, Thy-1.1+ and Thy-1.2+ congenic mice were used to determine whether Ts cells originate in the lymphoid tissue of the donor or recipient mice. In addition, T cell lines and clones were developed from cells that copurified with hapten-bearing APC from UV-irradiated and unirradiated animals to examine the mechanism of suppression. The present study examines the phenotype, pattern of cytokine production, and in vivo suppressor activity of cell lines and clones from unirradiated and UV-irradiated, hapten-sensitized mice. The influence of Ts cells on Th1 cell activation was also assessed by measuring the effect of Ts on APC functions, such as IL-12 production and activation of cloned Th1 cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female C3H/HeN (MTV-) and C57BL/6 mice were purchased from the animal production area of Frederick Cancer Research Facility (Frederick, MD). B6.PL-Thy-1a/CY congenic mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were maintained in a pathogen-free barrier facility in accordance with National Institutes of Health and American Association for Assessment and Accreditation of Laboratory Animal Care International Guidelines. The animals were housed in filter-protected cages and provided with National Institutes of Health open formula mouse chow and sterile water ad libitum. All procedures were approved by Institutional Animal Care and Use Committee. Each experiment was performed with age-matched mice between 10 and 12 wk of age.
UV radiation
UV radiation was provided by a bank of six unfiltered FS40 sunlamps (National Biologic, Twinsburg, OH). Approximately 65% of the energy emitted from these lamps is within the UVB range (280-320 nm), and the peak emission is at 313 nm. The average irradiance of the source was approximately 5 W/m2 at a 20-cm distance, as measured by an IL1700 radiometer with an SEE240 detector fitted with an SES280 filter and a W quartz diffuser (International Light, Newburyport, MA). Groups of five mice were anesthetized with nembutal (sodium pentobarbital, 0.01 ml/g body weight) injected i.p. and subsequently irradiated on shaved ventral skin. Mice received a single treatment of 2 kJ/m2 UVB. The ears of mice were protected from UV radiation by covering them with opaque tape. Control mice were treated in an identical manner, but were not exposed to UV radiation.
Contact hypersensitivity
UV-irradiated and unirradiated control mice were sensitized on shaved abdominal skin by applying 400 µl of 0.5% FITC (Isomer I; Molecular Probes, Eugene, OR) in acetone/dibutylphthalate (1:1 v/v). Mice exposed to 2 kJ/m2 UV radiation were sensitized through the irradiated skin 3 days later. Six days after sensitization, the mice were challenged by painting 5 µl of 0.5% FITC on the dorsal and ventral surfaces of each ear. Ear thickness was measured immediately before challenge and again 24 h later using a micrometer (Mitutoyo, Tokyo, Japan). Specific ear swelling was determined by subtracting the background swelling exhibited by mice that were challenged but not sensitized.
Cell transfer
Unirradiated and UV-irradiated mice were sacrificed 18 to 24 h after sensitization with FITC; their inguinal, brachial, and axillary nodes were removed, and single-cell suspensions were prepared from them. The cells were washed three times in RPMI 1640 medium containing 5% FCS, and 1 x 106 cells were injected into each hind footpad of recipient animals. On day 7 after sensitization, spleens were removed from those recipient mice that exhibited a decreased CHS response and from appropriate control animals. Single-cell suspensions were prepared by teasing the spleens apart in HBSS and filtering them through a nylon mesh. The cells were washed once and refiltered, and 108 viable nucleated cells were injected i.v. into syngeneic recipients. Immediately after injection, the recipients were sensitized with FITC, as described above.
Cell separation
Rat anti-mouse IgG2a-coated Dynabead M450 beads (Dynal, Great Neck, NY) were treated with anti-murine Thy-1.1 mAbs, according to the manufacturer’s instructions. Before spleen cell transfer, cell suspensions were enriched for T lymphocytes using a nylon wool column. The nonadherent, T cell-enriched fraction was incubated with the anti-Thy-1.1 Ab-coated magnetic beads in a slow rotator at 4°C for 45 min. At the end of the incubation, the cells were separated using a Dynal Magnet, and the supernatant containing unbound cells was collected. The cell-bound beads were washed twice and incubated at 37°C overnight in a humidified incubator to permit the detachment of the cells. The cells were washed twice, counted, and injected i.v. into congenic C57BL/6 mice.
Generation and FACS analysis of FITC-specific T cell lines
Unirradiated and UV-irradiated mice were killed 18 h after
sensitization with FITC, and a single-cell suspension was made from
their lymph nodes, as described above. The cell suspensions were washed
three times in RPMI 1640/5% FCS at 4°C and separated on 18%
metrizamide gradients (19). The cells at the gradient interface were
collected, washed, and sorted based on their forward scatter and FITC
content, using a Coulter Epics Elite cell sorter (Coulter, Hialeah,
FL). The cells were cultured for 2 wk in 96-well flat-bottom microtiter
plates (Costar, Cambridge, MA) at 1 to 2 x 105
FITC+ cells/well in a total volume of 200 µl RPMI 1640
medium containing 10% FCS (CELLect Gold; ICN Biochemicals, Aurora,
OH), 4 mM L-glutamine, 100 µg/ml penicillin, 100 U/ml
streptomycin, 10 mM HEPES, and 5 x 10-5 M
ß-mercaptoethanol (complete medium). After the initial 2 wk of
culture, the T cells were collected, washed, and added to 24-well
microtiter plates (Costar) at a concentration of 5 x
105 T cells/well with 1 to 2 x 106
-irradiated (2500 rad) metrizamide-purified APC from unirradiated
and UV-irradiated, FITC-sensitized syngeneic mice in a total volume of
1.5 ml. After 3 days, 1 ml of culture supernatant was removed and
replaced with fresh medium containing 3 U/ml murine rIL-2 (Boehringer
Mannheim, Indianapolis, IN). The cultures were fed every 3 to 4 days
with fresh medium containing 5 U/ml rIL-2, and kept at a concentration
of 5 x 105 to 1 x 106 cells/well.
The cultures were restimulated every 2 wk. The phenotype of the
lymphocytes was determined by staining the cells with
phycoerythrin-coupled Abs against CD4, CD8, TCR-ß, and TCR Vß4,
7, 8, and 11 (PharMingen, San Diego, CA), as follows: Cells were
incubated on ice for 30 min with 1 µg Ab/106 cells,
washed three times in PBS containing 1% FCS, and analyzed by flow
cytometry.
Proliferation assay
Lymph node APC from unirradiated, FITC-sensitized mice were isolated and purified, as described above, and cocultured with the T cell lines in medium containing RPMI 1640, 10% FCS, 2 mM L-glutamine, 5 x 10-5 M ß-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES buffer in 96-well U-bottomed microtiter plates (Costar) in a total volume of 200 µl/well. Cultures were incubated at 37°C in a 7.5% humidified CO2/balanced air atmosphere. After 24 h, 100 µl of culture supernatant was removed from each well for cytokine measurements. Fresh medium was added (100 µl/well), and the incubation was continued for 2 days. Cultures were pulsed with [3H]thymidine (2 Ci/mmol, 1 µCi/well; ICN Biomedicals) during the final 18 h of incubation. The cells were harvested onto glass filter fiber sheets using an automatic harvester (Tomtech, Orange, CT). Proliferation was assessed by [3H]thymidine uptake using a liquid scintillation counter (Betaplate; Pharmacia, Turku, Finland).
Cytokine analysis
Murine IL-10 was measured using Abs and rIL-10 purchased from
PharMingen. The cytokines IL-4 and IFN- were measured by ELISA using
commercially available kits (Endogen, Cambridge, MA). Biologically
active TGF-ß was measured using a Predicta TGF-ß1 kit
from Genzyme Diagnostics (Cambridge, MA), All assays were performed
according to the manufacturer’s instructions.
In vivo activity of T cell lines
The T cell lines were collected from culture and washed three times in RPMI 1640. The cells were counted, and their viability was assessed by trypan blue dye exclusion, and 5 x 103 to 5 x 105 cells were injected into the hind footpads of syngeneic recipient mice. The animals were sensitized on their shaved abdomens with 400 µl of 0.5% FITC and on their back skin with 50 µl of 0.3% 2,4-dinitrofluorobenzene (DNFB) in acetone. Six days after sensitization, the animals were challenged with 5 µl of 0.2% DNFB (Aldrich Chemical, Milwaukee, WI) applied to dorsal and ventral surfaces of one ear, and 5 µl of FITC applied to the dorsal and ventral surfaces of the other ear. The immune responses to the haptens were measured as described above.
Statistics
Statistical analyses were performed using two-tailed Student’s
t tests or ANOVA. A p value of 
0.05 was
considered significant. Analyses were performed using Statview SE +
Graphics software (Abacus Concepts, Berkeley, CA). For CHS experiments,
each experimental group contained at least five mice. All experiments
were performed at least twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfer of DLN cells from mice sensitized through UV-irradiated
skin results in the formation of Ag-specific Ts cells in the spleens of
the syngeneic recipients (8, 10). We investigated whether these
suppressor cells were descendants of the donor DLN cells or originated
in the recipients by using animals that were congenic at the Thy-1
locus. Thy-1.1+B6.PL-Thy-1a/Cy mice were
exposed to 2 kJ/m2 UVB, or not irradiated and sensitized
with FITC 3 days later. Their DLN were removed 18 h after
sensitization, and cell suspensions were injected into
Thy-1.2+C57BL/6 congenic recipients. Cells from
unirradiated mice produced a CHS response to FITC in the recipients
(Table I
, Expt. A), whereas cells from
UV-irradiated mice produced a significantly lower CHS response.
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, Expt. B). To determine whether the splenic Ts cells originated in the lymph nodes of the UV-irradiated donor mice, Thy-1.1+ cells were separated from Thy-1.2+ cells in spleen cell suspensions from the lymph node cell recipients. The cells were transferred to Thy-1.2+C57BL/6 mice that were then sensitized with FITC. Mice that received spleen cells from animals given DLN cells from unirradiated, FITC-sensitized donors had a CHS response similar to the sensitized control group. The CHS response was reduced significantly in mice given unseparated spleen cells from recipients of DLN cells from UV-irradiated, FITC-sensitized donors. Depletion of Thy-1.1+ cells from the spleen cell preparations of these mice resulted in the loss of Ts activity. The Thy-1.1+ cells, detached from the Dynabeads and injected into mice, significantly suppressed the induction of a CHS response to FITC. Transfer of as few as 3 x 105 Thy-1.1+ cells resulted in significant suppression of CHS responses (p < 0.05). These results demonstrate that the Ts were Thy-1.1+, and therefore, originated in the DLN of UV-irradiated donors.
Establishment of T cell lines
Because the Ts precursor cells are adherent to
FITC+APC in the DLN of UV-irradiated mice, we were able to
isolate these cells by FACS purification of the FITC+APC
and established cell lines (11). APC from the lymph nodes of
unirradiated and UV-irradiated, FITC-sensitized C3H/HeN mice or
DNFB-sensitized animals were purified on metrizamide gradients. The
large cells from all groups were selected by flow cytometry and
analyzed for fluorescence (Fig. 1). Two
populations of FITC+ cells were isolated from lymph node
cells of unirradiated and UV-irradiated, FITC-sensitized mice based on
their fluorescence content. Cells with low fluorescence intensity,
FITClow from unirradiated (Fig. 1A),
UV-irradiated (Fig. 1C), FITC-sensitized mice, and those
with high fluorescent intensity, FITChigh (Fig. 1, B and D, respectively) were isolated from the
same pool of lymph node cells. Approximately 15% of the cells in the
FITClow population were FITC-APC, based upon
the background fluorescence values provided by the cells from the
DNFB-sensitized animals. Cell lines were established by biweekly
restimulation with IL-2 and -irradiated,
metrizamide-purified homologous APC from either unirradiated
or UV-irradiated, FITC-sensitized mice.
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Two cell lines, TUVhigh11/8/4 and
TUVhigh2/21/5, were independently derived from
lymphocytes adherent to FITChighAPC from UV-irradiated,
FITC-sensitized mice. Another cell line,
TUVlow2/21/5, was derived from the same pool of
unseparated lymph node cells that was used to isolate the
TUVhigh2/21/5 line, but was adherent to
FITClowAPC. Two months after isolation, the Ag specificity
and suppressor activity of the cell lines were determined in vivo by
injection of 5 x 104 cultured T cells in the hind
footpads of syngeneic mice. Control mice received normal syngeneic
spleen cells or saline. The mice were immediately sensitized with FITC
on their abdomen and DNFB on the back. Six days after sensitization,
CHS responses to the haptens were evaluated by challenging the animals
with FITC on one ear and DNFB on the other. Animals sensitized in this
manner developed significant CHS responses to both haptens, as measured
by ear swelling 24 h after challenge (Table II). The reduction of CHS responses to
FITC and DNFB observed in mice given syngeneic spleen cells from
untreated animals was not statistically significant. Injection of the
TNRhigh2/21/5 cell line neither suppressed nor
augmented the CHS response to the homologous hapten. The response of
those same animals to an unrelated hapten, DNFB, appeared to be
enhanced over the unimmunized control group; however, the effect was
not statistically significant (p > 0.05). Injection
of the T cell lines TUVhigh11/8/4 and
TUVhigh2/21/5 significantly
(p = 0.01, <0.001, respectively) suppressed
the induction of CHS responses to FITC. The suppression was specific
for the homologous hapten since there was no effect on the response to
DNFB. In contrast, TUVlow2/21/5 cells failed to
suppress CHS responses to either hapten. The experiments were repeated
once with the TUVhigh11/8/4 cells and three times
using the TNRhigh2/21/5 and
TUVlow2/21/5 cell lines with similar results.
These data are consistent with our previously published work on the in
vivo activity of the lymph node cell populations from which the T cell
lines were derived (11). The results demonstrate that both T cells and
FITC+APC from DLN of UV-irradiated, hapten-sensitized mice
are heterogeneous; APC contain different amounts of FITC and bind to
Ag-specific T cells that differ in their ability to suppress CHS
responses in vivo.
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The in vitro Ag specificity of the T cell lines was investigated
by culturing the cells with lymph node APC from mice sensitized with
FITC or the unrelated hapten DNFB, and spleen cells from untreated,
syngeneic mice coupled with FITC in vitro. The results, presented in
Figure 2, show that all of the T cell
lines from both unirradiated and UV-irradiated mice were specific for
the immunizing Ag; however, they failed to respond to spleen cells
conjugated with FITC in vitro. The failure of in vitro coupled spleen
cells to stimulate the T cell lines was not due to the source of APC
since identical results were obtained using lymph node cells from
unimmunized animals (not shown).
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Phenotype of the T cells
The profile of cytokines produced by the T cell lines from
unirradiated and UV-irradiated, FITC-sensitized mice was examined to
assess their helper phenotype. The cell lines that had been maintained
in vitro for 2 to 6 mo were cultured for 24 h with APC from the
DLN of FITC-sensitized mice. Culture supernatants were collected and
assayed for cytokines by ELISA. The T cells were also injected into
mice to determine their in vivo suppressor activity. The
TNRhigh2/21/5 cells, derived from unirradiated,
FITC-sensitized mice, were predominantly Th1 like, producing large
amounts of IFN- and TGF-ß, but only low levels of IL-4 (Table III).
TUVhigh2/21/5 cells from
UV-irradiated mice produced significantly higher quantities of IL-10
and IL-4 than the TNRhigh cells, but similar amounts
of IFN-, thereby resembling Th0 or Th3 cells. Neither the
TNRhigh nor the TUVlow cells were
immunosuppressive in vivo.
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). These cells were derived from the
same pool of unseparated lymph node cells as the
TUVlow2/21/5 line, but produced intermediate
amounts of IFN-, and high levels of IL-4, IL-10, and TGF-ß.
Another Ts cell line, TUVhigh3/36/6, was
independently derived and secreted low levels of IFN-, high levels
of IL-4, and intermediate amounts of IL-10. The differences in
cytokines observed with the various T cell lines were not due to the
source of APC since the same spectrum of cytokines was observed when
the T cell lines were stimulated with immobilized anti-CD3 Ab (not
shown). Because the Ts cells produced IFN- in addition to IL-4,
IL-10, and TGF-ß, their phenotype could not be resolved using
uncloned cell lines.
The characteristics of the suppressor cells were further investigated
using cloned Ts cell lines. Two clones, B4 and F4, from the
independently derived Ts cell line TUVhigh3/26/6,
produced only IFN- in response to FITC-bearing APC and were thus
designated Th1 cells. Clones D4 and D6 produced IL-10, but no
detectable IL-4 or IFN-. These cells also produce significant
amounts of IL-10 within the first 8 h of culture (not shown).
Injection of 4 x 104 D6, but not B4 or F4 cloned T
cells into normal syngeneic mice significantly (p <
0.001) suppressed the induction of CHS responses to FITC (Table III).
Suppressor activity was observed with as few as 5000 cloned D6 cells;
however, higher numbers of these cells did not further reduce the
immune response in three separate experiments (not shown).
To determine their surface phenotype, the T cells were stained with
phycoerythrin-labeled Abs against costimulatory and TCR-associated
molecules. The cell lines derived from both UV-irradiated and
unirradiated, FITC-sensitized mice were CD4+,
CD8-, CD28+ (not shown). All lines were also
TCR-ß+, indicating that the cells did not originate
from TCR-
+ dendritic epidermal T cells that reside in
skin. Further analysis of the cells’ Ag receptor revealed that the
cloned T cells were Vß4-, Vß7-,
Vß8.1/8.2-, Vß8.3-, and
Vß11-.
MHC restriction
The observation that the T cell lines and clones responded only to
Ag presented by in vivo sensitized APC and failed to respond to cells
coupled with FITC in vitro, led us to ask whether the T cells were MHC
restricted and whether the restriction was encoded by genes in the I-A
or I-E regions of the MHC. APC from unirradiated, FITC-sensitized mice
were cocultured with the T cells in the presence or absence of Ab
against I-Ak or I-Ek molecules. The results
presented in Figure 3 show that
anti-I-Ak Ab completely blocked the proliferation of
the Th1-like B4 cells, but not the D6 Ts cells. In contrast,
anti-I-Ek Ab reduced the stimulation of the T cell
clone D6 by 60%, but had no effect on the activation of B4 cells.
Taken together, these data indicate that the Ts clone D6 recognizes Ag
in the context of I-Ek molecules, but that Ag recognition
by the Th1 clone B4 is I-Ak restricted.
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T cell immunity to haptens is mediated by activation of the Th1 subset of lymphocytes by Ag-bearing APC (12, 14). The cytokine IL-12 is produced by Ag-stimulated APC and activates Th1 cells while suppressing Th2 cell functions (20, 21). Suppressor T cells have been postulated to block the activation of Th1 cells, either directly or through their action on APC (22, 23). To determine the effect of Ts on Th1 cells, cloned D6 Ts cells were cocultured with Th1+, FITC-specific, B4 lymphocytes in the presence of APC from unirradiated, FITC-sensitized mice. The proliferation and cytokine profiles of the cultures were measured as indicators of cell activation.
The data presented in Table IV show that,
in the absence of T cells, cultures of -irradiated APC from
FITC-sensitized mice produced high levels of IL-12. Addition of the
Th1-like B4 clone of lymphocytes to the APC resulted in increased
production of IL-12 and IFN- in these cultures. In contrast, the
addition of D6 Ts cells to APC resulted in an 80% reduction in IL-12
in the cultures, probably through the action of the IL-10 on APC
functions. The D6 suppressor clone produced 500 to 1000 pg/ml IL-10
during the first 8 h of culture (not shown). Despite the effect on
IL-12 levels, the FITC-bearing APC stimulated proliferation of and
IL-10 production by the D6 Ts cells, indicating that IL-12 is not
necessary for the activation of these T cells. Addition of Ts
lymphocytes to cultures of Th1 B4 cells and APC nearly eliminated the
production of IL-12 (21 pg/ml), reduced IFN- and IL-10 levels, and
appears to have blocked the proliferative response of the Th1 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using adoptive transfer models to establish the mechanism of immune
suppression, several laboratories have shown that the functions of APC
from UV-irradiated, hapten-sensitized mice were altered and that immune
suppression was due to -radiation-sensitive, Thy-1+ and
CD4+ cells (10). We have previously shown that the Ts in
the DLN of UV-irradiated, FITC-sensitized mice copurify with a brightly
fluorescent (FITChigh) subpopulation of APC, and that the T
cells, but not the APC, are responsible for the induction of
suppression when transferred to syngeneic recipients (11). However,
whether these T cells were precursors of Ts or induced Ts cells in the
recipient mice was unclear (25, 26, 27).
The present study used donors and recipient mice that genetically differed only at the Thy-1 locus to determine whether the Ts cells arose directly from the donor-derived cells or were generated in the recipient animal. The data show that the Thy-1.1+, but not the Thy-1.2+, cells found in the spleens of recipients of cells from UV-irradiated mice suppressed the induction of a CHS response when transferred to syngeneic animals. These results demonstrate that the Ts arose from the DLN donors and did not induce a secondary Ts population in the recipients. Tokura et al. proposed that the spleens of UV-irradiated mice induced Ts when transferred to unirradiated recipients, and that the induction of the Ts cells could be blocked by cyclophosphamide treatment (26). While our data show that the Ts cells were not induced in the DLN cell recipients, the cells may suppress the immune response by the production of cytokines or other proteins (27, 28, 29, 30).
Whether the Ts cells originate in the skin or the periphery is unclear.
Hapten-derivatized epidermal cells from UV-irradiated skin induce
tolerance when injected into unirradiated recipients (31, 32). However,
all T cell lines derived in our study were
Thy-1+, CD4+, and
TCR-ß+, indicating that they arose from peripheral T
cells, and not from
Thy-1+CD3+I-Ak-CD4/CD8-TCR-+
dendritic epidermal T cells (33). Further evidence that Ts originate in
the DLN comes from Vink et al., who found cyclobutylpyrimidine dimers
in FITC-bearing lymph node APC, indicating that these cells had
migrated from the skin following UV irradiation and hapten
sensitization (34). Repair of the lesions in the DNA of the APC
restored immune functions and blocked the generation of Ts cells.
One mechanism proposed for the immunosuppressive effects of UV radiation is the differential activation of Th cell subsets by APC. APC exposed to UV radiation in vitro or from the spleens of mice systemically suppressed by UV radiation fail to stimulate Th1 clones, but activate Th2 cells normally (12, 13, 17). Several studies have suggested that Ts are Th2 cells that mediate the suppressive effects of UV radiation by down-regulating Th1 functions through their production of IL-10 (17, 18, 28, 29). Our results show that lymphocytes that copurified with FITChighAPC from unirradiated mice had a predominantly Th1 phenotype, consistent with the ability of the donor animals to respond to hapten. In contrast, T cells from UV-irradiated mice produced a broad spectrum of cytokines indicative of the presence of several T cell phenotypes (15). It was necessary, therefore, to establish cloned cell lines to clearly identify the Ts phenotype and to understand the mechanism of their suppressive activity.
The Ts cell lines D4 and D6 were cloned from lymphocytes that
copurified with FITChighAPC from UV-irradiated mice. These
cells produced IL-10 in the absence of IL-4 or IFN- and suppressed
CHS responses in vivo. The phenotype of our Ts clones resembles that of
Tr1 regulatory cells described by Groux et al. (35). Tr1 cells are
induced by chronic activation of lymphocytes in the presence of IL-10,
and are CD4+, ß+. They produced IL-10,
IFN-, and TGF-ß with little or no IL-2 and IL-4. The Tr1 cells
made early IL-10 and suppressed Ag-specific immune responses. We have
found that our suppressor clones also produced IL-10 within 8 h of
culture. However, unlike Tr1 cells, they failed to secrete IFN-.
Furthermore, our suppressor cell lines and clones did not exhibit
long-term stability, even when IL-12 was added to the cultures as a
growth factor. Finally, the Ts clones began to secrete IL-4 after
several months in culture and lost suppressor activity after 9 mo of
continuous maintenance in vitro (manuscript in preparation). Another
possibility is that our suppressor cells belong to the Th3 subset of T
lymphocytes, which also exhibit suppressor activity and can make
variable amounts of TGF-ß, IL-4, and IL-10 (36). The levels of IL-4
and IL-10 are linked in those cells. However, since our clones failed
to make IL-4 while still producing high levels of IL-10, we concluded
that they did not belong to the Th3 cell phenotype. Based upon these
analyses, we have tentatively designated our suppressor clones T
regulatory type 2 (Tr2). This phenotype may represent a distinct cell
type, be a subset of Tr1 cells, or be an intermediary state between Th0
and Th2. We favor the former hypothesis because, unlike either Th0 or
Th2 cells, our suppressor cells produce early IL-10 and change
phenotype only with repeated restimulation after many months in
culture. The failure to make IFN- and indifference to IL-12 as a
growth factor also favor a separate designation for these clones.
However, until the spectrum of cytokines associated with Tr1 cells is
more completely defined by the discovery of other cells of this type,
we cannot rule out that our Ts cells belong to this lymphocyte subset.
The presence of cytokines in the microenvironment can influence T cell maturation, and IL-10 appears to be necessary for the activation of Tr1 cells (35, 36, 37). Besides lymphocytes, keratinocytes and Mac1+APC are also a source of IL-10 in UV-irradiated skin (38, 39, 40). The finding that T cells produced IL-10 in the absence of IL-4 and were able to suppress CHS responses to hapten is consistent with a report by Yagi et al. (18), who showed that a tetrachlorosalicylanilide-specific Th2 Vß7+ cell line from UV-irradiated mice suppressed CHS responses through their production of IL-10, but not IL-4. Thus, the stimulation of IL-10 production appears to be a major mechanism by which UV radiation induces tolerance in vivo. The role of TGF-ß in mediating suppression in this system is unknown at present. Although this cytokine has been shown to be an important mediator of immune suppression in other models, the production of comparable amounts of biologically active TGF-ß by our nonsuppressive cell line and suppressor cells alike led us to conclude that another cytokine such as IL-10 may play a more important role in this model (41). However, an interaction between TGF-ß and IL-10 cannot be ruled out. The exact role of TGF-ß as a mediator of immune suppression in this model is being investigated in our laboratory.
UV radiation-induced Ts cells suppress the proliferation of
primed, Ag-specific T cells in culture, but the activity of the T cells
can be restored by addition of anti-IL-10 Ab (18). IL-12 acts as an
antagonist to IL-10 by preferentially stimulating Th1 cells and
inhibiting Th2 cells and their cytokines through a IFN--dependent
mechanism (20, 21, 42). Injection of IL-12 blocks the action of
preformed Ts cells and restores CHS responses (23). We found that IL-12
was produced by APC from the DLN of FITC-sensitized mice, and its
production was enhanced by the addition of IFN--secreting Th1 cells
to the culture (Table IV). In contrast, addition of the Ts cells nearly
eliminated IL-12 production, even in the presence of the Th1 cells. The
Ts cells reduced Th1 cell activation probably through the action of
IL-10 on APC functions (17, 29).
While IL-10 is important for suppression, T cells may suppress immune
responses by other mechanisms. The finding that FITClow T
cells from UV-irradiated mice also make high levels of IL-10, but
consistently fail to induce suppression in vivo, argues against IL-10
as the sole mediator. Kuchroo et al. have reported that soluble
-chain of TCR-ß+, I-E-restricted Ts hybridomas is
responsible for the function of these cells (27). T cells can also
reduce immune responses by inducing apoptosis in Ag-bearing APC (43, 44). We are currently investigating the role of apoptosis in the Ts
activity.
The observation that Ts copurified only with UV-FITChighAPC suggests that these APC may express altered or unique costimulatory molecules that enable the differentiation of naive, Ag-specific T cells along the Ts pathway. In vitro UV irradiation of APC has been reported to decrease the expression of MHC, adhesion, and costimulatory molecules on these cells (38, 45, 46). However, we have not observed a decrease in ICAM-1 expression and found only a slight reduction of B7-1 and B7-2 proteins on FITC+APC from UV-irradiated mice (11). Furthermore, the Ts cells preferentially responded in vitro to Ag presented by APC from unirradiated, FITC-sensitized mice compared with APC from UV-irradiated animals.
The T cells were both MHC restricted and required in vivo acquisition of the Ag, indicating that the TCR binds to a region of the hapten that couples to protein and includes conjugated amino acid residues (47). T cells that recognize this region of the FITC molecule, known as the protein proximal region, are MHC restricted and hapten specific. The spectrum of proteins that predominate in skin is different from those normally found on cell membranes. Inclusion of sequences from skin proteins such as keratin, as part of the T cell epitope, might account for the failure of the cells to recognize FITC coupled to APC in vitro. Finally, the amounts and distribution of FITC in APC coupled with the Ag in vitro are very different from the pattern observed on cells acquiring FITC in vivo (48). The quantities of Ag available on the APC indicate a fundamental difference in the way Ag is processed and presented by these two cell populations, and might account for differences in Ag recognition by the T cells.
In conclusion, cutaneous exposure to UV radiation results in the
activation of TCR-/ß+, Ts precursor cells in lymph
nodes draining the irradiated, hapten-sensitized skin that give rise to
Ts in the spleen. The Ts precursor cells adhere to a subset of
Ag-bearing lymph node APC, and produce IL-10, but not IL-4 or IFN-,
and may belong to a distinct T cell subset, Tr2. Suppression of CHS
responses by these cells might occur by reducing IL-12 production and
the stimulation of Th1 cells by APC.
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Acknowledgments |
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Footnotes |
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2 Authors V.K.S. and M.W.P. contributed equally to this work.
3 Current address: Wyeth Lederle Vaccines and Pediatrics Inc., Department of Viral Immunology-876, Pearl River, NY.
4 Address correspondence and reprint requests to Dr. Faith M. Strickland, Department of Immunology-178, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. E-mail address:
5 Abbreviations used in this paper: CHS, contact hypersensitivity; DLN, draining lymph node(s); DNFB, 2,4-dinitrofluorobenzene; Tr, T regulatory; Ts, suppressor T cells; UVB, ultraviolet B (280–320 nm) radiation.
Received for publication November 16, 1997. Accepted for publication April 8, 1998.
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ß heterodimer of the TCR. J. Immunol. 152:3.[Abstract]
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