The role of suppressor factors in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes

121,74-87 (1989)

CELLULARIMMUNOLOGY

The Role of Suppressor Factors in the Regulation of Immune Responses by Ultraviolet Radiation-Induced Suppressor T Lymphocytes I. Activity of Suppressor Cell Culture Supernatants’ GENE

K. YEE,*,~ STEPHEN E. ULLRICH, AND MARGARET L. KRIPKE

Department oflmmunology, University of Texas M. D. Anderson Cancer Center, Houston Texas, 77030 Received November I, 1988; accepted February 15, 1989 The purpose of this study was to determine whether soluble suppressor factors are involved in the regulation of immune responsesby ultraviolet radiation-induced suppressor T lymphocytes (UV Ts). The UV Ts were induced by applying contact allergens to the ventral, unirradiated skin of mice that had been exposed 5 days earlier to UVB radiation. Supernatants from cultures that contained a mixture of UV Ts, normal responder lymphocytes, and hapten-mcdified stimulator cells were injected iv into normal recipients at the time of sensitization; they inhibited the induction of contact hypersensitivity (CHS) in vivo in an hapten-specific manner. The supematants similarly suppressedthe generation of specific cytotoxic T lymphocytes (CTL) in vitro. Moreover, supernatants from cultures that contained either UV Ts alone or UV Ts in combination with either the responder or the stimulator cells failed to suppress the CHS and CTL responses.These results suggestthat hapten-specific inhibitory factors may participate in the regulation of immune responsesby suppressorcells generated by epicutaneous sensitization of UV-irradiated mice. o 1989 Academic PRSS, Inc.

INTRODUCTION Ultraviolet (UV) radiation is a ubiquitous environmental carcinogen and the primary cause of human skin cancer (1). In addition to its carcinogenic activity, UV radiation induces a selective, systemic alteration in the immune system that contributes to the growth of skin cancer (2, 3). Experiments in mice have revealed that this alteration involves the generation of tumor-specific suppressor T lymphocytes that prevent the rejection of UV-induced tumors (2,4,5). One approach used to investigate how UV radiation activates the suppressor cell pathway is based on the finding that antigen-specific suppressor T lymphocytes (UV ’ This work was supported by Grant RR55 I l-22 from the National Institutes of Health, Grant 83-191 from the Sid Richardson Foundation, and funds from the M. D. Anderson Annual Campaign, a project of the University of Texas Cancer Board of Directors. ’ A Rosalie B. Hite Fellow of the University of Texas, M. D. Anderson Cancer Center. 3Present Address: Department of Pathology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118-2394. 74 0008-8749/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form resewed.

SUPPRESSOR FACTORS FROM UV Ts CULTURES

75

Tsr are induced in UV-irradiated mice after epicutaneous application of a contact allergen to the unirradiated skin (6). The suppressor cells generated in this model of UV-induced systemic unresponsiveness inhibit a contact hypersensitivity (CHS) response against the sensitizing antigen. They resemble the tumor-specific suppressor cells in that they act on the afferent arm of the immune response, and they have the surface phenotype Thy-l+, Lyt-l+, Lyt-2- (7). Recent studies have indicated that, in addition to inhibiting the CHS response, UV Ts of this phenotype can suppress the proliferation of normal lymphocytes (8) the generation of cytotoxic T lymphocytes (CTL) to hapten-modified syngeneic cells in vitro ((9), unpublished data), and the production of antibodies in vivo against the trinitrophenyl hapten (9). Other recent data from our laboratory have indicated that the suppressor cells exert their suppressive effect by blocking the proliferation or activity of hapten-specific helper T lymphocytes (Th) in the cell-mediated response ( lo- 12). The mechanisms by which UV Ts block the proliferative response of Th is not known. One possibility is by the production of soluble suppressor factors (TsF) by the UV Ts. Such factors have been demonstrated in other suppressor cell systems (reviewed in ( 13- 16)). Moreover, the involvement of TsF in local UVB-induced unresponsiveness has been reported recently ( 17, 18); in those systems, the antigens were administered directly on the irradiated site. Tokura et al. ( 17) have demonstrated a role for TsF in the suppression in vivo of photoallergy against tetrachlorosalicylanilide (TCSA) plus UVA radiation, and Aurelian et al. ( 18) have shown TsF to be involved in the suppression in vitro of the lymphoproliferative responseto herpes simplex virus type 2 (HSV-2)-infected cells. To examine the involvement of inhibitory factors in the function of UV Ts, supernatants were harvested from cultures containing UV Ts, normal responder lymphocytes, and antigen-coupled syngeneic stimulator cells and used as a putative source of TsF. The supernatants were examined for their ability to suppress the induction of CHS in vivo and the generation of CTL in vitro in a manner similar to that observed for the UV Ts. In the accompanying paper (39), cell-free lysates from sonically disrupted suppressor cells were used as a second source of TsF, and these were tested similarly for their ability to inhibit such responses. MATERIALS AND METHODS Mice. Specific pathogen-free female C3H/HeN (MTV) mice were supplied by the Animal Production Area of the Frederick Cancer Research Center. The animals were between 10 and 12 weeks old at the beginning of each experiment. Induction and elicitation of CHS. Either 100 ~1 of a 3% (w/v) solution of 2,4,6trinitrochlorobenzene (TNCB; Pfaltz and Bauer, Inc., Waterbury, CT) in acetone or 50 ~1 of a 0.3% solution (v/v) of 2,4-dinitrofluorobenzene (DNFB; Sigma, St. Louis, MO) was used as the sensitizing antigen. The contact allergen was applied to the shaved abdominal skin of the mice. Six days later, the ear thickness of each mouse was measured with an engineer’s micrometer (Swiss Precision Instruments, Los An4 Abbreviations used: CHS, contact hypersensitivity; CTL, cytolytic T lymphocytes; DNBS, 2,4-dinitrobenezenesulfonic acid; DNFB, 2,4-dinitrofluorobenzene; E:T ratio, effector-to-target ratio; HSV-2, herpes simplex virus type-2; NSC, normal spleen cell; NSC (DNP), dinitrophenyl-modified normal spleen cell; NSC (TNP), trinitrophenyl-modified normal spleen cell; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNCB, 2,4,6-trinitrochlorobenzene; UV Ts, ultraviolet radiation-induced suppressor T cells.

76

YEE, ULLRICH,

AND

KRIPKE

geles,CA), and each ear surface was painted with 5 ~1of the relevant antigen. Either a 1%solution of TNCB or a 0.2% solution of DNFB was used for challenge. One day later, the ear thickness was remeasured; the specific ear swelling was determined by subtracting the swelling produced by the challenge antigen on the ears of unsensitized mice (6, 19). Generation of suppressor T lymphocytes. The protocol used to generate suppressor T lymphocytes (UV Ts) has been described in detail previously (6). Briefly, the shaved dorsal skin of mice was exposed to a single 3-hr dose (40 kJ/m2) of UVB radiation (280-320 nm) from a bank of Westinghouse FS-40 sunlamps. During the irradiation, the ears of the mice were shielded with electrical tape. Five days after irradiation, the mice were sensitized by epicutaneous application of a contact allergen to their shaved, unirradiated abdominal skin, as described above. Six days later the mice were challenged, and CHS was measured 24 hr later. Control mice were similarly sensitized and challenged, but they were not irradiated (NR). Additional controls included mice that were exposed to UV radiation but not sensitized (UV-only) and untreated mice (normal). The spleens from mice that exhibited a suppressed CHS response (UVBirradiated, sensitized mice) were removed, single-cell suspensionswere prepared, and the red blood cells were subjected to osmotic lysis in water. In order to enrich for T lymphocytes, the cells were incubated on nylon wool columns and then eluted (20). Spleen cells from the NR, UV-only, and normal groups of mice were treated similarly and used as a source of control cells. Generation of hapten-specljk cytotoxic T lymphocytes. A modification of the procedure of Shearer (9, 2 1) was used in these studies. Normal spleen cells (NSC, 1 X 10’ cells/ml) were suspended in a 40 pg/ml solution of mitomycin C (Sigma, St. Louis, MO) in Hanks’ balanced salt solution (HBSS) and incubated in the dark for 30 min in a 37°C water bath. The cells were washed three times in HBSS and resuspended in HBSS, and an equal volume of a 20 mM solution of either 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) or 2,4-dinitrobenzenesulfonic acid (DNBS; Sigma) in HBSS (pH 7.4) was added. The cells were then incubated for 20 min as described above, washed three times, and used asa source of stimulator cells in the CTL culture. Stimulator cells (5 X 106) were cultured for 5 days at 37°C in a 5% CO2 plus air atmosphere with an equal number of NSC responder cells in RPM1 1640 medium that was supplemented with 10% fetal calf serum, 5 X lop5 M 2-mercaptoethanol, 100 units/ml penicillin, 100 hg/ml streptomycin, 2 mM L-glutamine, 1% sodium pyruvate, 10 mA4 Hepes buffer, MEM vitamins, and 1% nonessential amino acids (GIBCO, Grand Island, NY). The responder and stimulator cells were suspended in a total volume of 2 ml in each well of a 24-well culture dish (Costar, Cambridge, MA). The cultures were then harvested, and the CTL activity was assessedwith a shortterm, 4-hr 5’Cr-release assayagainst hapten-modified, 5’Cr-labeled target cells. Cells from a syngeneic, methylcholanthrene-induced fibrosarcoma (MCA- 113) were labeled with the relevant hapten and used as the target cells. Various numbers of effector cells were then cultured with 1 X lo4 TNP- or DNP-modified, “Cr-labeled target cells in 96-well round-bottomed microtiter plates (Costar, Cambridge, MA), yielding effector-to-target cell (E:T) ratios of 12.5:1, 25: 1, and 50: 1 in 200 &well of supplemented medium. The plates were spun for 8 min at 2508, incubated at 37°C for 4 hr, and respun prior to harvesting. One hundred microliters of supernatant was removed from each well, and the amount of radioactivity released by the target cells was measured in a gamma 5500 counter (Beckman, Fullerton, CA). The percentage cytotoxicity exhibited by each effector cell group was calculated by

SUPPRESSOR

percentage cytotoxicity =

FACTORS

FROM

UV Ts CULTURES

77

experimental release - spontaneous release x 100. total release - spontaneous release

The total release was measured in supernatants from wells of target cells alone to which a 1% solution of T&on-X had been added. The spontaneous releasewas measured from wells that contained target cells alone in supplemented medium. Production of suppressorfactors in UV Ts culture supernatants. Supernatants from 5-day-old cultures that contained a mixture of UV Ts, normal responder lymphocytes, and hapten-modified syngeneic stimulator cells were used as a source of TsF. Five million responder cells, 5 X lo6 stimulator cells, and 1 X lo7 UV Ts were incubated at 37°C for 5 days. Supernatants were harvested from the cultures by centrifugation at 25Og for 8 min. The supernatants were then spun for 12 min at 400g and passedthrough a 0.45~pm filter (Millipore, Bedford, MA). In these experiments, the supernatants were tested immediately for activity in vivo or in vitro. Control supematants were obtained from cultures that contained 5 X lo6 responder and 5 X lo6 stimulator cells alone, or cultures containing stimulator and responder cells plus 1O7 NR cells, lo7 UV-only cells, or 1O7normal cells. Assessment of suppressive activity in vivo. To determine the ability of the culture supernatants to suppressCHS, they were injected iv into normal mice prior to sensitization with the relevant contact allergen. In most experiments, 0.75 ml of the supernatants was injected. The recipient mice were then sensitized for CHS and challenged, as described above. Suppression of CHS was determined by comparing the responsesof sensitized mice with those of mice injected with supematant before sensitization according to percentage suppression = 1 -

specific ear swelling of test group x 100. specific ear swelling of control group

Assessment of suppressive activity in vitro. To test the suppressive activity of the culture supematants in vitro, they were added to new cultures of NSC responder and hapten-modified stimulator cells in volumes (0.5 ml) that constituted 25% ofthe total volume of the CTL culture. These cultures were incubated for 5 days, and the CTL activity was assessedin a 4-hr “Cr-release assay against hapten-modified MCA- 113 target cells, as described above. Protein content analyses. The microassay protocol of the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA) was used to determine the protein content of the supematants, according to the manufacturer’s instructions. The absorbance values of the samples were measured at 600 nm by a Titertek Multiskan Model 3 10 (Flow Laboratories, Inc., McClean, VA). Statistical analyses. The two-tailed Student t test was used to assessthe significance of differences in CHS and CTL activities. The CTL data were converted into a normal distribution by the arcsine transformation (22) before analysis with Student’s t test. RESULTS SuppressiveActivity of the Culture Supernatants To determine whether the supernatants harvested from cultures of UV Ts, responder, and stimulator cells contained suppressive activity, various amounts were injected into normal mice at the time of contact sensitization with TNCB. Only the injection of supematants derived from cultures of responder NSC and stimulator

78

YEE. ULLRICH, Group -

Cells Added

Hapten

ml of factor injected

1

NO cells

none

None

2

NO cells

TNCB

NCHE

3

U”Ts

TNCB

0 10

AND KRIPKE

0.25 0 50 0.75 1 .oo

4

NR

TM%

0 10 0 25 0.50 0.75 1.00

5

W-Only

TNCB

0.75

6

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0.75

7

No cells

TWB

0.75

F I 0

I

I

I

10 Ear Swelling

I

20 t

so

(ml

x 10

-3

)

FIG. 1. Suppression ofCHS by UV Ts culture supernatants. Responder NSC (5 X lo6 cells) were cultured with an equal number of mitomycin C-treated, TNP-modified stimulator cells. Suppressor cells (UV Ts) were generated in UV-irradiated, TNCB-sensitized mice, and the UV Ts were added to the cultures, as described. The culture supernatants were injected iv into normal recipients in volumes ranging from 0.1 to 1.Oml. Control supernatants included those derived from responder and stimulator cells alone (group 7) and those to which control cells were added (groups 4-6).

cells [NSC (TNP)] plus the added TNP-specific UV Ts [UV Ts (TNP)] suppressed CHS (Fig. 1, group 3). When this group (stipled bars) was compared to the positive CHS control (group 2), significant suppression (P < 0.001) was observed following the injections of 0.5 to 1.0 ml of supernatant. Neither the supernatants harvested from cultures of responder and stimulator cells alone (group 7) nor those from cultures containing these cells and the added control regulator cells (groups 4-6) significantly inhibited CHS compared to those from the positive response (group 2, P > 0.5). Thus, only supernatants from cultures containing UV Ts, plus the stimulator and responder cells, were capable of suppressing the CHS response to TNCB. A volume of 0.75 ml of supernatant (corresponding to 104 pg total protein) was utilized in the subsequent CHS experiments. Requirements for the Generation of Suppressor Factors To determine the cellular requirements for the elaboration of TsF into the culture supernatants, TNP-specific UV Ts were cultured alone, in the presence of either responder or TNP-modified stimulator lymphocytes, or in combination with both cell types. The supernatants from these cultures were then assessedfor their ability to suppress CHS induction in vivo and CTL generation in vitro, as described above. In studies involving CHS, the supematants were injected into mice at the time of

79

SUPPRESSOR FACTORS FROM UV Ts CULTURES Group Responder

Stimulator

Regulator -

-

Haplen

NO supernatants

inpcled

r&w

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injected

TNCE

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None

UVTs

TNCB

ra

NO”=

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TNCB

tt32

NSC (TNP)

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TNCB

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NOM

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TNCB

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(TNP)

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k

i-

F

0

I

I

I

I

I

5

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2s

Ear Swelling

t_ SD (cm. x 10. 3,

FIG. 2. Requirements for the generation of TsF that suppress CHS. Supematants derived from cultures of UV Ts alone (group 3), UV Ts with either NSC responders (group 4) or TNP-modified stimulators (group 5), or UV Ts in combination with both cell types (group 6) were injected iv (0.75 ml) into normal mice. The mice were sensitized with TNCB and challenged after 6 days, and CHS was assessed.Control supernatants that were harvested from the cultures of similar combinations of cells but with the substitution of NR control cells for the UV Ts were similarly utilized (groups 7- 10). In addition, the results obtained with supematants from cultures that contained only responder and TNP-modified stimulator cells are represented by group 11.

sensitization with TNCB (Fig. 2). Mice that received supernatants from the cultures containing UV Ts, responder, and stimulator cells exhibited a decreased CHS response (group 6) when compared with the positive TNCB control (group 2, P < 0.001). In contrast, supernatants from cultures containing UV Ts alone (group 3) or those that also included responder or TNP-modified stimulator cells (groups 4 and 5, respectively) did not suppressthe CHS response to TNCB. In addition, none of the control supernatants derived from cultures that included NR cells alone, NR cells with either responder or stimulator cells, or NR cells in combination with both inhibited the CHS response (groups 7- 10). Supernatants from cultures that contained only responder and stimulator cells similarly failed to inhibit this response (group 11). Thus, a combination of UV Ts, normal responder lymphocytes, and antigen-modified stimulator cells is necessary for the elaboration of TsF into the culture supernatants. The second test for TsF generation was performed using the in vitro CTL assay (Fig. 3). Supernatants were added at the time of culture initiation to responder and TNP-modified stimulator lymphocytes. The generation of CTL was then assessed after 5 days with a CTL assay against TNP-modified MCA- 113 target cells. The response obtained in CTL cultures to which supernatants derived from cultures con-

80

YEE, ULLRICH, AND KRIPKE Group

Supernalant Responder ~~

1

Source

Stimulator

No supernalants

Regulator

added

2

None

NO%

uv Ts

3

Nsc

NC%

UV Ts

4

None

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5

NSG

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m

None

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None

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(TNP)

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9

hi32

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(TNP)

NR

I

I-

t-

0

I

I

I

I

1

1

I

10

20

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40

50

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Percent

Specific

Lysis of TNP-m&fled

Target

Cells

FIG. 3. Requirements for the generation of TsF that suppress CTL generation. Supematants derived from cultures of UV Ts alone (group 2) UV Ts with either NSC responders (group 3) or TNP-modified stimulators (group 4), or in combination with both cell types (group 5) were added to CTL cultures at the time of initiation in a volume (0.5 ml) that represented 25% of the total volume. Five days later, the CTL activity was assessedwith a 4-hr “Cr-release assay against TNP-modified MCA- 113 target cells. Control supernatants harvested from the cultures of similar combinations of cehs, with the substitution of NR control cells for the UV Ts, were similarly utilized (groups 6-9).

taining UV Ts; responder, and stimulator cells were added (group 5) was significantly decreased when compared with the positive CTL response obtained in cultures to which no supernatants were added (group 1, P < 0.001). As was the case with the CHS experiments described above, supernatants from cultures that contained UV Ts alone or UV Ts plus either responder or stimulator cells did not inhibit the CTL response (groups 2-4). Similarly, the supernatants from control cultures did not suppressthe CTL response(groups 6-9). Taken in combination with the results obtained in the CHS experiments, these results indicate that interactions between UV Ts and both normal lymphocytes and hapten-modified stimulator cells must occur in order for TsF to be produced.

Kinetics of the Generationof SuppressorFactors To determine the kinetics of TsF generation in the culture supernatants, supernatants from cultures containing DNP-specific UV Ts or control NR cells, normal responder lymphocytes, and DNP-modified stimulator cells were harvested at various times following the initiation of culture. The supematants were injected iv into normal mice at the time of contact sensitization with DNFR (Fig. 4). Mice that received supematants from cultures containing UV Ts harvested 3 days following initiation exhibited a significantly decreasedCHS responsewhen compared to the positive control for CHS against the contact allergen (P < 0.001). The maximum suppressive activity was detected by Day 4 in supematants harvested from cultures containing

SUPPRESSOR FACTORS FROM UV Ts CULTURES

81

UV Ts supematant NR supernatant

Days following

culture

initiation

FIG. 4. Kinetics of the generation of TsF. Supematants from the cultures of UV Ts, NSC responder, and DNP-modified stimulator cells were harvested at various times following the initiation of culture and injected iv (0.75 ml) into normal mice. The mice were sensitized with DNFB and challenged after 6 days, and CHS was assessed.Control supematants from cultures containing NR cells in lieu of UV Ts were harvested at the corresponding time points following culture initiation and were similarly utilized.

UV Ts, with no significant change in the level of suppressive activity by Day 5. In contrast, supernatants from control cultures taken at the corresponding time points did not significantly suppress the CHS response. These results indicate that the interactions among the UV Ts, responder, and hapten-modified stimulator cells result in detectable TsF activity in the supernatants within 3 days following culture initiation, with the peak in suppressive activity occurring within 4 days. Specificity of the UV Ts Culture Supernatants in Vivo We next investigated the antigenic specificity of the suppression induced by the supernatants. Both TNP-specific UV Ts and DNP-specific UV Ts were generated. These suppressor cells were then cultured with normal responder lymphocytes and stimulator cells that were modified with the relevant hapten. The supematants harvested from these cultures were injected into normal recipients, which were then sensitized with the same or a different hapten to assessthe specificity of the suppression. Of the recipients that were sensitized with TNCB (Fig. 5), only those that received iv injections of supernatants from cultures of responder, TNP-modified stimulators, and TNP-specific UV Ts exhibited a decreasedCHS response (group 3) compared to the positive TNCB control (group 2, P < 0.001). In contrast, the supematants that were derived from cultures of responder cells, DNP-modified stimulator cells, and DNP-specific UV Ts did not suppressthe CHS response to TNCB (group 8). In addition, neither the supernatants from cultures containing responder and stimulator cells alone (group 7) nor those with the added control cells suppressed the CHS response (groups 4-6). Thus, only supematants derived from cultures containing TNPspecific suppressor cells could suppressthe CHS response against TNP. A similar pattern was observed in the reciprocal experiment with DNFB sensitization (Fig. 6). Only mice that received supernatants from cultures of responder, DNPmodified stimulator, and DNP-specific UV Ts exhibited a depressedCHS response (group 3) when compared to the positive DNFB control (group 2, P < 0.00 1). Supernatants derived from cultures of responder cells, TNP-modified stimulator cells, and TNP-specific UV Ts did not suppress the CHS response to DNFB (group 8). Taken together, these results indicated that the suppressive activity present in the culture supematants is hapten-specific when tested by CHS in vivo.

82

YEE, ULLRICH, supernatant

Group Responder

t@k”

source

Stimulator -

AND KRIPKE

Regulator I

NO superna,an,s

Injected

None

NO supernatants

mjected

TNCB

(TNP)

U”

Ts (TNP)

TNCB

Nsc

WC

EC

NSC (TNP)

NR (TNP)

TNCB

EZ

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TNC?

Nsc

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NX

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None

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UV TS (DNP)

TNCB

MX

NSC (DNP)

NR (DNP)

TN%

(TNP)

t-

I 0

I

I

I

Ear Swelling

I

t

SJ

(ml

I 30

20

10

x 10. 3,

FIG. 5. Specificity of UV Ts culture supernatants in the suppression of CHS against TNCB. Culture supematants were prepared as described, and 0.75 ml was injected into normal recipients. The mice were sensitized with TNCB, challenged, and examined for a CHS response.In addition to the supematants from the cultures of responder, TNP-modified stimulators, and TNP-specific UV Ts (group 3) or its associated control cells (groups 4-6) supematants from cultures of responder and stimulator cells alone are included (group 7). Supernatants derived from cultures with responder, DNP-modified stimulator cells, and DNPspecific UV Ts are represented by group 8 and those with its associatedcontrol by group 9.

Specificity of the UV Ts Culture Supernatants in Vitro The second test of hapten specificity was performed using the in vitro CTL assay. It was shown previously that the addition of UV Ts to CTL cultures suppressedthe generation of CTL in an antigen-specific manner (9). To determine the specificity of the supernatants in suppressing CTL generation, supernatants from 5-day cultures containing responder, stimulator, and suppressor or control cells were tested for their ability to inhibit the generation of primary CTL. The supernatants were added to cultures of responder NSC and antigen-modified stimulator cells. Five days later, the generation of hapten-specific CTL was assessedin a CTL assay against target cells conjugated with the corresponding hapten (Fig. 7). Only supernatants from cultures that initially contained responder NSC, TNP-modified stimulator cells, and UV Ts (TNP) suppressedthe generation of TNP-specific CTL (group 2). A 75% suppression of CTL activity compared to that of the controls (P < 0.001) was obtained when the suppressive supernatant was added. Similarly, only supernatants from cultures initially containing responder NSC, DNP-modified stimulator cells, and UV Ts (DNP) suppressedthe generation of DNP-specific CTL (Fig. 8, group 2). The percentagesuppression observed was 64% when compared to that of the controls (P < 0.00 1). These results indicated that not only was the magnitude of suppression obtained with these supernatants similar to that obtained with the suppressor cells ((9), unpublished data), but more important, hapten-specific suppressor factors found in the UV Ts

83

SUPPRESSOR FACTORS FROM UV Ts CULTURES supernatant

Group Responder ---

Hapten

source

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No supernatants

injecled

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DNFS

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b&2

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EC

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!-

I 0

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I

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Ear Swelling

I

I

I 30

20

t_ XI

(cm

x 10

-3

)

FIG. 6. Specificity of UV Ts culture supematants in the suppression of CHS against DNFB. Culture supematants were prepared as described, and 0.75 ml was injected into normal recipients. The mice were sensitized with DNFB, challenged, and examined for a CHS response.In addition to the supematants from the cultures of responder, DNP-modified stimulators, and DNP-specific UV Ts (group 3) or its associated control cells (groups 4-6), supematants from cultures of responder and stimulator cells alone are included (group 7). Supematants derived from cultures with responder, TNP-modified stimulators, and TNP-specific UV Ts are represented by group 8 and those with its associated control by group 9.

culture supernatants were capable of suppressing the generation of CTL in vitro and CHS in vivo. DISCUSSION The purpose of the studies presented here (and in the accompanying manuscript) was to investigate the involvement of TsF in the regulation of immune responses by UV Ts. In this report, supernatants derived from cultures of UV Ts, normal responder, and hapten-modified stimulator lymphocytes were used as a putative source of TsF, and they were tested for suppression of CHS responses in vivo and CTL generation in vitro. The results imply that UV Ts may suppress these responses through the elaboration of TsF. Initial studies on the specificity of UV Ts in the suppression of CHS (6) demonstrated that UV Ts generated in TNCB-sensitized mice inhibited CHS against TNCB, but not DNFB. In the studies presented here, the specificity of suppression by the UV Ts culture supernatants mirrored that of the suppressor cell population. The suppression induced by the supematants was specific for the hapten used to generate the UV Ts. The specificity of the supernatants was also demonstrated using the CTL assay. It is interesting to note that we failed to detect suppressive activity in the supematants of cultures of UV Ts alone or UV Ts plus either responder or antigen-modified

84

YEE, ULLRICH, Group -

Supernatant -Responder

1

AND KRIPKE

Source

Stimulator -

No supematants added

Regulator

++

FIG. 7. Specific suppression by UV Ts culture supernatants of the generation of TNP-specific CTL. Responder NSCs (5 X lo6 cells) were cultured with an equal number of mitomycin C-treated, TNP-modified stimulator cells. Supematants harvested from suppressedcultures that contained responder, haptenmodified stimulator cells, and either TNP- or DNP-specific UV Ts or their corresponding control cells were then added in a volume (0.5 ml) that constituted 25% of the total volume in the CTL cultures. Five days later, the generation of CTL was assessedby a 4-hr 5’Cr-release assay against TNP-modified MCA113 target cells. The results from a E:T ratio of 50: 1 are shown.

stimulator cells. Culturing suppressor cells alone has previously been demonstrated to be sufficient to produce TsF. In the DNFB system, Claman et al. (23) obtained TsF from 24-hr cultures of lymph node cells from mice injected iv with DNBS and subsequently sensitized in viva with DNFB. These TsF were antigen-specific and suppressedonly the efferent, or elicitation, arm of the CHS response. Factors possessing the same characteristics were demonstrated by Zembala and Asherson (24) following a similar protocol using TNBS and TNCB. Although the antigens used in these systems are identical to those used in our study, the protocol for inducing Ts and the source of the Ts are clearly different, and the period of incubation necessary for the production of TsF is different. In particular, we did not give a second in viva sensitization with hapten after induction of the Ts. Our requirement for interaction of UV Ts, responder, and stimulator cells in vitro may be equivalent to the second in vivo immunization in the studies ofclaman et al. (23). These differences may also account for the fact that our culture supernatants, like the UV Ts, suppress the induction of the CHS response. The requirement for UV Ts plus a normal responder cell and a hapten-modified cell to produce TsF is reminiscent of another TNCB system (25,26). Following production of TsF-producing cells by the above-mentioned procedures, culture of the cells produces TsF that can interact with normal macrophages which in turn produce nonspecific suppressor factors. The requirement for such a normal cell is similar to our system; however, the resulting TsF in our system is hapten-specific and requires the presence of hapten. Zembala and colleagues (27, 28) demonstrated the presence of a “T acceptor cell” (Tact) from lymph node and spleen cells. This cell interacts with TsF, and following triggering by hapten-modified spleen cells, it releasesnonspecific inhibitory factors. This TsF acts on an efferent-acting Ts that releasesthe penulti-

85

SUPPRESSOR FACTORS FROM UV Ts CULTURES Group

supernatant Responder

1

Source

Stimulator -

NO supernatants

-Regulator

added

2

m

NSC (DNP)

3

m

NSC (DNP)

NR (DNP)

4

m

NSC (TNP)

UV TS (TNP)

5

Nsc

NSC (TNP)

NR (TNP)

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Nsc

NSC (DNP)

UV-OlllY

7

F6c

NSC (DNP)

Normal

UV Ts (DNP)

t-

1 rr

0

5 Percent

Speciffc

10 Lysis of DNP-modified

15 Target

Cells

FIG. 8. Specific suppression by UV Ts culture supematants of the generation of DNP-specific CTL. Responder NSCs (5 X lo6 cells) were cultured with an equal number of mitomycin C-treated, DNP-modified stimulator cells. Supematants harvested from suppressedcultures that contained responder, haptenmodified stimulator cells, and either DNP- or TNP-specific UV Ts or their corresponding control cells were then added in a volume (0.5 ml) that constituted 25% of the total volume in the CTL cultures. Five days later, the generation of CTL was assessedby a 4-hr “Cr-release assay against DNP-modified MCA113target cells. The results from an E:T ratio of 50: 1 are shown.

mate nonspecific factor that inhibits the effector T cell of CHS. The Tact is not necessarily a normal cell, however, since immunization is required for its production. In our studies, it is not clear whether the UV Ts is itself the producer of TsF or the inducer of the cell that does. Alternatively, both of the above mechanisms may act simultaneously, resulting in the production of TsF that suppress multiple antigenspecific immune responses.The suppressive factors found in the supernatants of UV Ts, responder, and antigen-modified stimulator cell cultures thus differ from those described above in their specificity, ability to suppress the induction of CHS, and their requirements for generation. It is not known whether the UV Ts are capable of inducing other Ts in a cascadeas Claman et al. (23) and others ((25-28) reviewed in ( 13- 16)) have described. However, the UV Ts phenotype Thy- 1+, Lyt- 1+, Lyt-2-, the ability to suppress the induction of the response, and antigen-specific TsF resemble the characteristics of “first-order” suppressor cells described in various systems.These comparisons suggest that UV radiation induces either an earlier step in the same pathway described above or a unique Ts pathway. The experiments presented here indicate that TsF activity does not result from endogenous lymphocyte inhibitory factors or free antigens; generation of such activity requires cellular and kinetic requirements. Moreover, suppressive activity is hapten-specific, and none was detected in control supernatants. Moreover, they support the hypothesis that two distinct signals (7) are necessaryfor the induction of UV Tsmediated suppression. In this manner, the UV Ts are clearly distinct from Ts induced by other protocols. For example, in a number of systems that utilize similar antigens (23-28), introduction of the antigen iv is postulated to be sufficient for Ts induction, while subsequent sensitization triggers the production of TsF (23). It has been demonstrated previously (6) that irradiation with UVB alone is insufficient to produce Ts.

86

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AND KRIPKE

In addition to being a novel means of inducing Ts, UV radiation may induce unique classesof Ts, depending on whether the hapten is applied at a distant site (systemic) or through the UV-irradiated skin (local). Recent reports by Aurelian et al. ( 18) and Yasumoto et al. (29) provide useful basesof comparison between Ts and TsF that are involved in either local or systemic UV-induced suppression. In this system, mice were irradiated with UVB on the abdomen and infected with HSV-2 by intradermal inoculation at the site of irradiation (18, 29). The antigen-specific LytI’, L3T4+ population suppressed proliferative responses in viva, while nonspecific Lyt-2+ Ts were induced following exposure of splenocytes to HSV-2 ( 18). Supematants derived from splenocytes cultured for 4 days in the presence of HSV-2 antigens nonspecifically suppressed lymphoproliferation in vitro against HSV-2 (29). Upon chromatography on Sephadex, both antigen-specific and nonspecific fractions were detected, with the specific fraction being identified as a 115-kDa protein consisting of two disulfide-linked 70- and 52-kDa components ( 18). It is presumed that because splenocytes from HSV-Zimmunized mice were used as a source of TsF-producing cells, Ts, responder cells, and the equivalent of antigen-modified stimulator lymphocytes would be present in these cultures. Clearly, the kinetics of TsF production are not markedly different from our results, since in our system TsF production was first detected after 3 days of culture and reached a peak by Day 4. An obvious difference in the TsF produced in this system and in the data reported here is the lack of apparent specificity in the biological assay of the supernatants prior to fractionation. The nature of the antigen itself may account for this difference. It was reported that supematants derived from the cultures of splenocytes from nonirradiated mice plus HSV-2 antigen contained nonsuppressive activity ( 18). Alternatively, the protocol for generating Ts to HSV-2 is markedly different in that the antigen is administered at the site of UVB irradiation (local suppression), rather than at an unirradiated site (systemic suppression) as was performed in our studies. These differences in UV-induced suppression point out that UV radiation may induce unique classesof Ts, the particular one depending on whether local or systemic suppression is induced. The local suppression of the immune responsethat occurs when antigen is applied to UV-irradiated skin has been hypothesized to be the result of alterations in antigen presentation. Changes in Langerhans cell density at the site of exposure (30) and activation of a UV-resistant antigen-presenting cell population (3 I-34) may then result in the induction of Ts. In contrast, the distant suppression of CHS by UV Ts does not appear to be the result of either alterations in the number of Langerhans cells at the sites of irradiation (35) or in numbers and activity of such cells at the unirradiated site of antigen sensitization (35-37). Moreover, the reconstitution of intact antigen-presenting cells in UVB-irradiated mice did not reverse the effects of distant suppression. In light of these differences, it would not be surprising that these two types of Ts exhibited alternate mechanisms of suppression, in this caseby producing different types of TsF. It should also be noted that Tokura and colleagues (17) recently demonstrated the presence of specific TsF in the lysates of splenocytes that had been subjected to repeated cycles of freezing and thawing. The splenocytes were taken from mice exposed to suberythemal doses of UVB and then sensitized on the irradiated site by TCSA plus UVA radiation. This protocol and the results are discussedin more detail in the accompanying report. The results from the studies that are presented here indicate that antigen-specific inhibitory factors may be involved in the regulation of the immune response by UV Ts. It also may be hypothesized that UV radiation induces a unique class of Ts com-

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