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The influence of ultraviolet light irradiation on the immune system
CLINICAL
IMMUNOLOGY
AND
17, 141-
IMMUNOPATHOLOGY
155 (1980)
REVIEW The Influence
IRA J. Fox, LINDA
of Ultraviolet Light Irradiation Immune System’ L. PERRY, MAN-SUN
SY, BARUJ BENACERRAF, I. GREENE
MARK Department
on the
oj’ Pathology, Harvard Medical Boston, Massachusetts Received
March
School, 02115
25 Shattuck
AND
Street,
13, 1980
INTRODUCTION
Ultraviolet (uv) light irradiation has been associated with the development of sarcomas and carcinomas (1). This phenomenon has been best studied in mice, but similar effects occur in humans (2). Recently, the “escape” of uv-induced tumors has been shown to be related to the influence of ultraviolet light on the immune system (1, 3). The immune response to antigens reflects the participation of many elements of the immune system. Thymus-derived (T), bone marrow-derived (B), and antigenpresenting (APC) cells (probably of monocytic origin) are required for many humoral responses (4). Furthermore, cellular reactivity of T cells reflects the interactions of T-cell subsets defined by distinct differentiation antigens of the Lyt series (5). Thus, Lyt 1+23- T helper cells can augment target cell lytic responses mediated by Lyt l-23+ T cytotoxic cells (6). Delayed-type hypersensitivity (DTH) reactivity is mediated by Lyt l+ T cells, although synergistic interactions with other T-cell subsets such as Lyt 123+ cells may also be required for maximal responsiveness (6,7). Cells which proliferate in vitro in response to antigen-pulsed APC are Lyt l+ cells. The interaction and clonal expansion of these T-cell subsets after exposure to antigen is subject to regulation by T suppressor cells (Ts). Ts function is complex and in some experimental systems is dependent upon at least two interacting subsets of suppressor cells (8). Antigen activates first-order Ts termed Ts, which release soluble suppressor factors (TsF) that induce secondorder T cells (Ts,) to limit immune reactivity (8). In this review, we shall provide evidence that a critical cellular component necessary for immune responses is uniquely impaired by uv irradiation. It has also been possible, employing uv treatment, to elucidate multiple levels of antigen recognition which could serve as targets for immune regulation. We will then consider the possible immunological mechanisms involved in uv-induced tumor enhancement and discuss how the use of uv irradiation might provide a practical approach to graft rejection. ’ Supported
by Grants
CA-14732
and AI-16396. 141 0090-1229/80/090141-15$01.00/O Copyright All rights
0 1980 by Acadermc Press, Inc. of reproduction m any form reserved
142
FOX
EFFECT
OF ULTRAVIOLET
ET AL.
IRRADIATION
OF CARCINOGENESIS
Tumor induction as a consequence of ultraviolet (uv) light irradiation has been studied in humans and in experimental animal models (1, 2). Photobiologists have found that uv-induced tumor development reflects a variety of cellular alterations associated with treatment with particular spectra of uv light. These may include the photochemical conversion of cell-bound steroids into carcinogenic compounds (9, lo), the release of liposomal hydolases as stimulators of inflammation and damage to essential cellular proteins (11, 12), and the cross-linking and dimerization of host cell DNA (reviewed in (12)). The protective capacity of ingested antioxidants has suggested a role for dietary factors in uv carcinogenesis as well (12, 13). With the possible exception of damage and/or faulty repair of uv-treated DNA, experimental evidence to support these proposed mechanisms of photocarcinogenesis is lacking, however (12). More recently, the relationship between uv light treatment and the expression of host cell-mediated immunity has been investigated. These productive studies have shed some light on the cellular basis of susceptibility to tumor growth in uv-treated animals. Tumors induced in mice by prolonged exposure to uv light irradiation display a high degree of antigenicity. Such tumors are indeed frequently rejected upon transplant to normal syngeneic recipients (14) (see Fig. 1). The antigens responsible for tumor rejection appear to be largely tumor specific, since normal animals immunized against a particular uv-induced fibrosarcoma and then subjected to sublethal X irradiation are capable of rejecting only the immunizing tumor upon subsequent rechallenge (14). The antigenic properties of uv tumors are similar to those of chemically induced neoplasms, in that tumors developing as a result of treatment with agents such as methylcholanthrene (MCA) generally display unique tumor-specific transplantation antigens (TSTA) even when tumors arise in the same inbred strain or host (15, 16). There is increasing evidence to indicate, however, that the antigens expressed by MCA-induced tumors may contain common or cross-reactive determinants as well as unique TSTA. In vitro (17) proliferation assays and in vivo transplantation studies (18) have demonstrated common antigens among tumors. The presence of cross-reactive antigens has also been suggested in the case of uv-induced tumors, by serological analysis (19), and by tumor transplantation. Thus, mice hyperimmunized against a particular uvinduced fibrosarcoma by multiple injections of tumor cells may reject other uvinduced tumors to which they did not previously respond. Precise definition of the determinants contributing to tumor resistance in the uv system is lacking, therefore, and may involve a variety of antigenic target structures. Resistance against uv-induced tumor transplants appears to be mediated primarily by cellular rather than humoral immune mechanisms. For example, animals which have been rendered immunodeficient by adult thymectomy and X irradiation (20) or by treatment with cyclophosphamide (21) or anti-lymphocyte serum (21) demonstrate a reduced in vivo response against the tumor. Normal responsiveness can be restored to such animals by the adoptive transfer of lymphocytes from normal or tumor-immune donors (14, 22). The subsets of cells responsible for the protection have been recently shown to be T cells by Daynes and colleagues (23).
ULTRAVIOLET
INFLUENCE
ON
. 2~10~1316 I” Normal C3H 0 2x to6 1316 in UV Treated C3H . 2 x i06 I316 in Immune UV Treated
THE
IMMUNE
SYSTEM
143
Mice
DAYS
FIG. 1. uv-Induced tumor growth in uv-treated, normal or immune uv-treated mice. C3H uv-treated mice were prepared for use by Dr. M. Kripke and were challenged with 2 x 10” 1316 fibrosarcoma (tumor also supplied by Dr. Kripke). Seven days later the tumor was removed and 28 days later these mice were rechallenged with the relevant tumor. Normal C3H, or uv-treated C3H, were also challenged at that time with 2 x lob: 1316. Tumor growth was followed daily. Whereas normal or immune uv-treated mice rejected tumor, uv-treated mice did not. It should be noted, however, that many weeks (>6) had elapsed between the time the uv-immune group were last treated with uv irradiation and the experiment depicted above.
In contrast to the spontaneous rejection response of normal syngeneic hosts, animals previously treated with subcarcinogenic levels of uv irradiation demonstrate progressive tumor growth when challenged with uv-induced antigenic tumor cells (20). The dosage of irradiation necessary to induce tumors varies between individual tumor lines and inbred host strains; nude or albino mice requiring less exposure than agouti or black animals (24). In certain cases, this effect can be accomplished with a single prolonged uv light treatment (26), suggesting that total dose rather than duration of treatment may be the determining factor (26). Definition of the cellular basis of the defective anti-tumor response in the uv-treated host has been approached by examining multiple parameters of T-cell and macrophage activation, in vivo as well as in vitro. Results have shown that although uv-treated mice are incapable of rejecting syngeneic uv-induced tumor grafts, their response against H-2 incompatible tumor allografts and H-2 allogeneic or semisyngeneic skin grafts appears normal (20). Other assays of immune function such as primary hemagglutinin responses to sheep erythrocytes and inflammatory reactions against dimethyl sulfoxide are also equivalent to those of untreated hosts (1,3). Analysis of the functional nonspecific reactivity in vitro of lymphoid cells from uv-treated mice has revealed normal patterns of responsiveness to concanavalin A and lipopolysaccharide and equivalent levels of macrophage tumoricidal and phagocytic capabilities (27). In vitro cytotoxicity against allogeneic or hapten-modified syngeneic cells (3) was also uninhibited, while, under conditions of a 48-hr microcytotoxicity assay, little or no cytotoxic activity
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ET AL.
was apparent against uv-induced tumor cells (28). The defect induced by uv treatment appears to be detectable only under specific conditions. Indeed, generally, the uv-treated host does not display an accelerated tumor growth rate when challenged with tumors induced by chemical carcinogens or other unrelated means (29). On the basis of these findings, it was suggested that uv treatment may induce a state of specific nonresponsiveness or tolerance to antigens expressed on uvinduced tumors (21, 30). Tolerance to an antigen has been shown to occur by at least two mechanisms, one involving the inactivation of T-cell clones specific for a given antigen by either clonal abortion, receptor blockade, or other ill-defined means (31), and another reflecting the generation of suppressor T cells (Ts) that have the capability to inhibit the expression of specific T-cell effector responses (32). Suppressor T cells have been shown to influence immunity to a variety of tumor types (33-35), such that Ts which arise during primary tumor development suppress the expression of T-cell immunity to tumor antigens, and, as a result, inhibit tumor rejection (33). Suppressor T cells in many systems are characterized by the expression of antigens encoded by the I-J subregion of the murine histocompatibility complex (MHC), and by the elaboration of I-J+ factors that participate in a suppressor cell circuit to induce secondary antigen-specific Ts (36). Depending on the system employed, Ts may suppress either the afferent (37) or the efferent (38) limb of a cellular immune response. Investigations into the possible contribution of suppressor cells in the enhancement of uv-induced tumors by previous uv irradiation revealed that these cells may also play a role in the proposed tolerance to uv tumor antigens (21, 30, 39). This has been demonstrated by a variety of means. First, it was found that tumor immunity transferrable to lethally X-irradiated recipients by normal syngeneic cells could be abrogated by the cotransfer of lymphoid cells from uv-irradiated mice (22,30). Second, susceptibility to uv-induced tumor growth could be conferred onto normal individuals by joining the latter in parabiosis with uv-treated animals (30). Finally, the transfer of spleen cells from uv-treated mice into normal recipients inhibited the normal animal’s capacity for rejection of uv-induced tumors (21). The cells responsible for this suppression were shown to be derived from the thymus (21) and sensitive to the effects of anti-Thy 1 antiserum plus complement (21), in accordance with the phenotype of Ts operative in other tumor systems (40). Interestingly, the presence of a growing tumor in uv-irradiated animals is not required for the generation of suppressor cell activity (21, 30). This observation raises some interesting and important questions regarding the mechanism of suppressor cell generation and the specificity of the reactive cell. According to current theories, Ts are stimulated by the presence of excess free antigen, such as that shed from the surface of live or disintegrated tumor cells (41). This hypothesis derived from the observations that Ts can be generated by antigen in macrophage-free cultures in vitro (41) and can bind to antigen-coated plates (42), indicating specificity for antigen alone. In the case of uv-induced Ts, it could be argued that cellular alterations that may accompany uv treatment, as the dimerization and faulty repair of host cell DNA, may result in the creation of new antigenic structures on the surface of affected cells similar to those expressed by uv-induced tumors. The steps leading from these changes to the stimulation of Ts
ULTRAVIOLET 100-
Bop
INFLUENCE
ON THE IMMUNE
SYSTEM
145
UV lrradloied C3H o 2x 10~ 1316 (H-29 + 21NMS/day .2 x106 1316 + Zl/day (~6~12 x BI~A (3R1) F, Ant!-BlOA (5R)
FIG. 2. uv-Treated C3H or normal C3H mice were challenged with 2 x 10” 1316 (H-2”) cells subcutaneously, on Day 0. Beginning at the time of tumor challenge, selected groups also received 2 PI/day/mouse of normal serum of 2 ~1 of anti-I-Jk antiserum (DBA12 X BIO.A(3R))F, anti-BlO.AtSR). Anti-I-Jk-treated mice had highly significantly reduced tumor growth.
could then be the result of failure to present antigen in the appropriate context of macrophage Ia determinants, which is normally required for the activation of positive effector T-cell activity (43). Evidence for the dysfunction of antigenpresenting cells in the uv-treated host will be presented in the following sections of this review. Transplantation of uv-induced tumors into the uv-treated host may stimulate additional suppressor cell activity, with specificity for antigens of the uv tumor. That these Ts contribute to progressive tumor growth in the uv host has been demonstrated after eliminating suppressor activity in viva. This has been accomplished by treatment of the uv-treated tumor-bearing host with alloantiserum specific for I-J subregion encoded determinants (44) using a protocol that has been shown to inhibit effectively Ts generated by chemically induced tumors (45) (see Fig. 2). Under these conditions, tumor growth in the antibody-treated host is significantly reduced and in some cases completely halted (44). Therefore, the failure of the uv-treated host to eliminate a tumor challenge successfully is probably (i) the result of the activation of multiple clones of antigen-specific Ts, including those induced by the tumor as well as those activated by the uv treatment itself, and (ii) the consequence of shared specificities for uv-induced antigens and uv-induced tumor cells. The underlying basis for Ts stimulation and for the apparently normal responses to mitogens and allogeneic stimuli will be elucidated further below. IN VITRO ANALYSIS OF THE CELLULAR EFFECTS OF ULTRAVIOLET IRRADIATION ON THE IMMUNE SYSTEM
The data strongly suggest that there exists a specific defect in immunity arising from irradiation with uv light and resulting in the stimulation of specific Ts. The
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ET AL.
precise mechanism by which this alteration occurs, however, has remained obscure. Several investigators have suggested that experiments using in viva models involve multiple, complicated mechanisms, all of which may not be controlled for within an experiment. In order to avoid the pitfalls associated with such work, these researchers have chosen to study the effects of uv light directly on cells involved in the immune system by in vitro analysis. In this regard, it has been found that lymphocytes are easily inactivated by ultraviolet light; their viability is compromised in vitro by as little as 100 mJ/cm2 (46). At uv doses which do not influence viability as judged by trypan blue exclusion (47, 48), these uv-treated lymphocytes nevertheless demonstrate little DNA synthesis in response to mitogens such as phytohemagglutinin (PHA) (47). Membrane properties such as the expression of alloantigen (determined by H-2 antibody absorption and cytotoxicity with complement) and the binding of PHA are apparently uninfluenced by uv irradiation (47, 49). In addition, irradiated cells do not appear to have acquired toxic properties. For example, it is well established that both free PHA and PHA bound to syngeneic lymphocytes stimulate responding cells to proliferate. It has been shown that uv-irradiated lymphocytes bearing PHA are equally efficient inducers in this system. It is also well established that if either prokaryotic or eukaryotic cells are irradiated directly with uv light, DNA replication is inhibited by the formation of pyrimidine dimers (50). Similar inhibition has been reported with respect to RNA and protein synthesis (46). Further, ultraviolet light treatment has been reported to cause induction, inactivation, or alteration in enzyme function (51). It is now known that these changes may reflect more than just the direct mutagenic effect of uv irradiation. Such changes in gene expression may, for example, be a cellular response to generalized stress induced by ultraviolet irradiation. Since different uv wavelengths elicit different biological effects, there is some confusion as to the nature of the effect which causes altered lymphocyte function; alterations generated by irradiation in the A region (320-400 nm) of uv, for example, appear to be oxygen dependent in certain systems, whereas C region (200-290 nm) uv light-induced damage is relatively oxygen independent (52, 53). Hence, the mechanism of A region-induced change must be different from that of C region-induced change. In addition, it has been demonstrated that uv irradiation of tissue culture medium containing cell nutrients can result in the death of cells growing in that medium (51, 54,55). Presumably, this is a result of the generation of toxic by-products. The consequences of such an effect on the study of immunocompetent cells are important since the presence of photoproducts in media may lead to misinterpretation of the effects of uv light on in vitro systems. Irradiation of cells in nonnutritive medium should eliminate such effects and exclude this potential artifact. THE INFLUENCE
OF ULTRAVIOLET
LIGHT ON ALLOREACTIVITY
It has been generally accepted that a difference in certain major histocompatibility complex (MHC)-encoded antigens on the cell membrane between a stimulator and a responder cell is critical for inducing proliferation in the mixed lymphocyte reaction (MLR) (56-58). To what extent these MHC-encoded anti-
ULTRAVIOLET
INFLUENCE
ON
THE
IMMUNE
SYSTEM
147
gens by themselves determine immunogenicity has never been fully defined. For example, A strain platelets, though carrying some of the same histocompatibility antigens as A strain lymphocytes, are incapable of inducing the same degree of MLR stimulation to B strain allogeneic cells (59). This observation raises doubt as to whether MHC antigen recognition alone accounts for MHC immune responsiveness. It has been suggested that only adherent cells are capable of acting as potent stimuli for the MLR in vitro. T cells or B cells, in the absence of adherent cells, provide little MLR stimulus to T-cell responders. It may be that allogeneic determinants might be presented to responder cells by antigen-presenting cells within the adherent cell population. Irradiation of immunocompetent allogeneic cells with uv light has been used to help determine which signals mediate antigenic-specific T-lymphocyte activation. Work in several laboratories has convincingly demonstrated that purified splenic T lymphocytes are unable to respond to uv-irradiated allogeneic cells in primary mixed lymphocyte and cytotoxicity assays (47, 51, 60, 61) (Fig. 3). In contrast, following in vitro mixed lymphocyte sensitization with normal cells, “secondary’‘-type mixed lymphocyte and cytotoxicity responses have been elicited by restimulation with uv-irradiated cells in both mouse and human systems (49, 60-64). However, if responding cells, in a primary stimulation, are confronted simultaneously with an ultraviolet-irradiated cell and a second cell differing only at the I region with the responding cell, a strong cytotoxic response to the K or D region of the uv-irradiated cell is elicited (61, 65, 66). Therefore, uv treatment seems to abrogate I region-associated stimuli required for activation of such immune cells. Although I region products appear to be affected, the specific defect induced by irradiation with ultraviolet light is at present unclear. There are several possible targets. Several investigators have demonstrated biologically potent growth factors, T-cell stimulation factors, and other products which are elaborated from cells of the immune system (67-69). The production of these products by either macrophages and/or lymphocytes may well be sensitive to uv I
I - 60,000
Stimulators
l 5xlO6/ml 0 1 x 106/ml
- 50,000 - 40,000
A 5 x 10s/ml
- 30,000 - 20,000
8 b P
- I0,000 -0 I1 0
1
IO T/ME
I
I
20 EXPOSED
II
I
30
I
40
I
I
50
TO UV LIGHT
I
I
60
I
fsecl
FIG. 3. The ability of uv light to interfere with MLR. A/J (H-29 mouse spleen cells were used as responders to mitomycin C-treated BALBic (H-2”) stimulator cells that had been exposed to uv light for the time periods indicated on the abscissa. The stimulator cells were splenic adherent cells that had been recovered from two cycles of adherence and separation from plastic plates. The uv light was delivered using a bank of FS40 sun lamp lights (2.0 J/m”/sec of 280-340-nm light).
148
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ET AL.
light. Alternatively, since antigenically stimulated cells make contact with other cells in culture by membrane extensions (70), uv-treated cells may have simply lost some aspect of their motility and, therefore, present antigen poorly. uv Irradiation may modify cell surface antigens and membranes, causing a loss or alteration of relevant molecules needed for immune responses. Indeed, following sensitization in viva with uv light-treated allogeneic lymphocytes, one can obtain very strong in vitro cytotoxicity responses to uv-treated cells (62). However, mice primed to untreated allogeneic cells respond in a limited manner to uv-treated allogeneic stimulators in vitro. The argument could be raised that by irradiating lymphocytes with uv light one induced a structural modification in the histocompatibility antigens. It is possible to propose a mechanism for T-cell activation from the above data. Bach and others have proposed a model to account for T-cell activation requiring two discrete signals (62). Signal 1 would be mediated by the antigenic target, which on the mouse cell would be either K or D region determinants. Signal 2, which is sensitive to uv irradiation and derived from I region-associated products, would, on the other hand, stimulate a separate population of proliferating helper cells which are necessary to maximize the response to K and D targets. This model should be extended to include antigen-presenting cells (APC), which are an integral part of T-cell activation. In this regard, it has been shown that membrane extracts and subcellular preparations will stimulate resting allogeneic lymphocytes in vitro if and only if a non-T-cell population is present in the responding population, presumably to present antigen (69, 71-80). Indeed, there is some evidence that the population of APC can be supplied by either the stimulating or responding population in alloreaction (74). It is likely, as will be discussed later, that the APC is the target of uv irradiation. There are, however, several inconsistencies in the literature with regard to work involving uv light. It has been reported that human sperm cells are not only capable of stimulating allogeneic lymphocytes, but that these cells are resistant to uv light when inducing primary allogeneic proliferative responses (81-84). Interestingly, sperm cell antigens cross-react with histocompatibility determinants on leukocytes but cannot restimulate their own primary sensitization, In vitro sensitization with sperm cells results in alloantigen-specific secondary MLR when leukocytes are used for restimulation but not when sperm cells are used for restimulation (84). Whether T-cell suppression of the sperm cell-induced immune reaction occurs when sperm cells are used to restimulate in the latter case is not known. However, there remains some doubt as to the general applicability of analyzing cell surface antigens of sperm to study alloreactivity or the effects of uv on the immune system. For example, it is well established that the inactivation of sperm DNA requires a higher dose of uv irradiation than does DNA from other tissues (85, 86). It should be noted as well that uv-induced changes in secondary MLR are also variable. Bach et al. could show that secondary responses are only inconsistently triggered by uv-treated cells, whereas others have found consistent restimulation (61). The explanation for these differences is not clear. It is known, however, that leukocytes treated with uv light and held overnight are much less efficient in hapten-presenting function to primed cells than leukocytes that are
ULTRAVIOLET
INFLUENCE
ON
THE
IMMUNE
SYSTEM
149
irradiated and added to culture within a few hours (84). Insufficient control of such factors may lead investigators to erroneous conclusion, and one can only postulate other as yet undefined variables associated with uv effects on cells. Whatever the mechanism, it is clear that some function (probably involving I-region-bearing cells, such as APCs) is sensitive to uv irradiation. This function is an essential component of the immune response, since, once ablated, effector immune reactions are not induced. In terms of recognizable effects on animals treated with uv irradiation, aside from the aforementioned tumor model, other work has consistently demonstrated that uv-treated animals possess little or no generalized alteration in immune function (27). Indeed, such animals, as previously mentioned, though unable to reject uv-induced syngeneic tumors, rejected both H-Zincompatible skin and tumor allografts (1). In addition to those studies described above, lymphocytes from animals receiving chronic ultraviolet irradiation in doses capable of inducing primary tumors maintained (i) normal plaque-forming cell response to PVP, (ii) normal primary cytotoxicity in vitro to non-uv-induced syngeneic tumors, and (iii) normal induction of local GVH reaction (normal animal recipients of lymphocytes from uv-irradiated donors) ( 1- 3). However, some alteration in the immune response of these animals has been demonstrated. Lymphocytes from irradiated animals display a 20-50% lower proliferative response to MLC stimulation than do lymphocytes from untreated animals (3). Similarly, uv-irradiated animals developed a transiently depressed GVH response to normal allogeneic cells during the early months of irradiation. In addition, delayed hypersensitivity to dinitrochlorobenzene (DNCB) was also found to be transiently depressed but returned to normal prior to the development of primary tumors (1, 5). Since the above changes in immunity were present only early during treatment and did not correspond to the development of tumors, there was some concern whether these mechanisms were operating in uv carcinogenesis. In vitro work was initiated to look for altered macrophage function since two of the alterations noted above primarily measured macrophage infiltration. Macrophages from uv-treated mice displayed (i) normal phagocytosis following opsinization, (ii) normal PEC induction in response to inflammatory agents, and (iii) normal in vitro tumoricidal capacity (27). In addition, spleens from irradiated animals contain a normal distribution of &bearing, immunoglobulin-bearing, and adherent cells when compared with spleens from normal animals. Thus, these studies were unsuccessful at localizing a defect. It was only when detailed in vivo studies using exogenous antigens were performed that a specific immunologic defect was identified that may explain, at least in part, uv-induced carcinogenesis. EFFECT
OF ULTRAVIOLET IRRADIATION HYPERSENSITIVITY
The first report ability to mount made by Haniszko of guinea pig skin
ON DELAYED-TYPE
indicating that uv-irradiated animals may be defective in their effective delayed-type hypersensitivity (DTH) responses was and Suskind (87). These investigators found that uv irradiation results in unresponsiveness to topical challenge with a contac-
150
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ET
AL.
tant, dinitrochlorobenzene (DNCB). However, if challenge was done at an unirradiated site on the same animal, the contactant elicited an apparently normal DTH response. Thus, the effect of limited uv-irradiation in the guinea pig appeared to be localized only in the exposed area. More recently, extensive studies on the effect of uv irradiation on DTH immunity in the murine system have been performed in the laboratory of Kripke and colleagues (1, 5, 14). Consistent with the above-mentioned, earlier findings, these investigators found that uv-treated mice displayed a transient unresponsiveness in DNCB contact sensitivity. This alteration lasted for the first 2 to 3 months of uv irradiation, after which these animals recovered normal responsiveness. The mechanisms by which uv irradiation brought about this transient reduction in DTH was not clear, as lymphocytes from uv-treated DNCB-primed mice could transfer immunity to naive recipients. Therefore, it was postulated that the de& ciency might reside in the earlier antigen-processing steps of the immune response . Definitive evidence of an antigen-presenting cell defect in uv-irradiated mice was provided in recent experiments by Greene et al. (88). These studies were made possible when it was found that a state of hapten-specific DTH could be induced in normal mice by the subcutaneous injection of hapten-conjugated syngeneic spleen cells. Fractionation of spleen cells according to their adherent properties on plastic petri dishes revealed that haptenated adherent cells were the most potent inducers of DTH as compared to similarly treated nonadherent cells. To examine the effect of uv irradiation on antigen-presenting cell function, normal and uv-treated BALB/c mice were immunized with TNP-conjugated normal adherent cells or TNP-conjugated adherent cells from uv-treated animals. The results, shown in Table 1, clearly demonstrate that normal animals develop significant DTH irrespective of the source of adherent cells. In contrast, uv-treated animals failed to produce significant levels of immunity when they were immunized with TNP-adherent cells obtained from uv-treated animals. This defect could be circumvented by immunization with TNP-derivatized adherent cells from normal mice, stimulating significant levels of DTH compatible to that of normal mice. From the experiments (depicted in Table 1, Experiment I), it was concluded that adherent cells from mice irradiated with uv light had lost their antigenpresenting cell function, and that the ability of TNP-conjugated uv-adherent cells to induce significant immunity in normal hosts was probably due to representation of antigen by the host antigen presenting cell. Most important was the finding (Table 1, Experiment II) that immunization of uv-treated mice with haptenderivatized cells obtained from uv-treated donors led to the generation of haptenspecific suppressor T cells. These Ts, upon adoptive transfer to normal recipients, abrogated their ability to respond to hapten-conjugated cells. Thus, uv light not only interferes with APC function but, as a consequence, predisposes the animal to the generation of Ts to any antigen which the APC would normally present. DISCUSSION
It has been difficult to determine whether uv irradiation directly alters host immunity. Multiple in vitro and in vivo assays of B-cell, T-cell, and phagocyte
uv uv -
IV V VI VII
TABLE
I
IO’ uv Tnp spl adh IO’ uv Tnp spl adh -
10’ uv Tnp spl adh IO’ uv Tnp spl adh
-
lo7 Tnp spl adh IO’ uv Tnp spl adh -
-
Immunizing cell”
-
5 X 10’ spleen cells
-
5 x IO7 spleen cells 5 x 10’ spleen cells (anti-Thy I.2 + C treated) 5 x IO’ spleen cells (NMS + C’)
-
-
5 X IO’ spleen cells 5 X IO7 spleen cells
-
Cells transferred to second recipient
7%, TNCB 0.5% DNFB 0.5% DNFB -
7% TNCB 7% TNCB 7% TNCB
7% TNCB 7% TNCB 7% TNCB -
1% TNCB 1% TNCB 1% TNCB
1% TNCB 1% TNCB
1%TNCB
1% TNCB
Challenge second recipient” -t k 5 k
5.0 4.0 4.0 3.0
26.0 85.0 86.0 6.0
‘f + -c
1.0 5.0 5.0 5.0
47.0 ? 5.0 27.0 -t 2.0 42.0 k 5.0
71.0 72.5 37.0 18.0
lo-” in ? SEM
APC LEADS TO THE
1% TNCB 0.2% DNFB 0.2% DNFB 0.2% DNFB
DERIVED
Immunization of second recipienY
WITH HAPTEN-DERIVATIZED W-TREATED APC BUT NOT NORMAL GENERATION OF SUPPRESSOR T CELLS
ns
-
P’
” Ultraviolet light irradiation of shaved mice for 1 hr. three times a week for I month using the FS40 sun lamp (2.0 J/m’/sec of 280-340-nm light). ” Adherent (spl adh) or nonadherent cells from the spleen (6) or peritoneal exudate cells (PEC) were derived with 10 m&f TNBS as described (688). ’ Immunization with 7% PCI (6) or 0.5% DNFB on the shaved abdomen as described previously (I). ” Challenge was performed using 1% TNCB in olive oil on the ear on Day 5. Measurements using an engineer’s caliper were performed 24 hr later. The increment of the challenged ear above the unchallenged was measured and recorded. (’ P values determined by the two-tailed Student’s I test.
uv
uv
uv -
uv
Expt II 1 II 111
Expt I I 11 III 1V
Group
Treatment of mice”
IMMUNIZATION
152
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ET AL.
function appear to be unaffected by uv light (1, 3, 14, 20-22). Several studies investigating antigen presentation to T cells in viva, however, suggest that uv irradiation does alter cellular immunity. Research in contact sensitivity and delayed-type hypersensitivity has confirmed such suspicions. In the systems under study, uv irradiation has resulted in a depressed DTH response, which is mediated by antigen-specific Ts. uv-Irradiated animals maintain normal T- and B-cell function, but are capable of mounting a normal immune response only if antigen is appropriately presented, for example, as seen in Table 1, when splenic adherent cells covalently coupled with hapten obtained from a normal animal are used for presentation. Splenic adherent cells obtained from uv-irradiated animals and then coupled with hapten are unable to induce a normal CS response in uv-irradiated hosts. Therefore, some aspect of antigen presentation is altered in these animals. Since cells within the spleen display this alteration, the ability to present antigen must be the function of a pool of cells which can be affected by nonpenetrating uv skin irradiation. It is likely that APCs function by recirculating between central and peripheral lymphoid compartments where they routinely encounter foreign antigens. uv Irradiation has been shown to decrease the number of I-A+ cells present at the site of irradiation. Therefore, if irradiating mice with uv light kills or alters APCs directly, the normal trafftc of such cells through the skin would eventually create an environment where APCs, once destroyed, could not be replaced. The mechanisms involved in Ts induction are not as yet delineated and are complex. If antigen is coupled to splenic adherent cells, and these cells are then injected iv into normal recipients, such animals generate antigen-specific Ts (37, 38). In at least one system, it appears that the ability to generate suppression is dependent upon APC presentation of antigen (44). However, in uv-irradiated animals such Ts seem to be generated as a consequence of impaired APC function. This mechanism may account for uv-induced photocarcinogenesis. Cellular alterations associated with uv treatment may result in the formation of new antigenic structures on the surface of affected cells; uv-induced tumors may develop tumor-specific antigens in just this manner. Since these antigenic structures are presented to the immune system by uv-impaired APCs, mechanisms similar to those found in contact sensitivity probably induce I-J+ Ts which are specific for the uv-induced neoantigens (44). Future work using the effects of uv light should help determine whether some special APC is needed for Ts activation. There is preliminary evidence indicating that the Langerhans cell, a type of monocyte found in the epidermis, is critical for inducing immunity in contact sensitivity. Investigators have noted a simple relationship between the density of Langerhans cells at the site of priming and the generation of immunity or suppression (89). Whether this implies that functionally different APC subsets modulate immune responsiveness is not yet known. The developmental maturity of APCs might, for example, play a pivotal role in the issue of immunity versus tolerance; or alternatively, entirely distinct APC cells may be responsible for these diverse effects. Direct uv irradiation of immunocompetent cells has also demonstrated that
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antigen recognition is mediated by at least two signals which result in immunity. One signal has been shown to be uv sensitive and is associated with cells bearing I-region gene products. Whether uv irradiation alters Ia surface antigens or affects the APC cell itself has not been determined. Work is presently under way to define what aspect of I-region-associated function is altered. In a similar manner, the uv system may help delineate the signals which function in the suppressor pathway. It is hoped that future work using uv light will continue to provide insight into the issue of how antigen processing relates to the generation of suppression and self-recognition. It may be possible to render grafts less susceptible to rejection, perhaps by uv irradiation of the recipient as well as the graft. Such treatment might result in the development of Ts specific for the relevant foreign antigens. At present experiments of this nature are underway in our laboratory. REFERENCES 1. Kripke, M. L., Lofgreen, J. S., Beard, J., Jessup, J. M., and Fisher, M. S., J. Nat. Cancer Inst. 59, 1227, 1977. 2. Marshall, V. C., Transplantation 17, 272, 1974. 3. Spellman, C. W., Woodward, J. G., and Daynes, R. A., Transplantation 24, 112, 1977. 4. Katz, D. H., and Benacerraf, B., Transplant. Rev. 22, 175, 1975. 5. Huber, B., Cantor, H., Shen, F. W., and Boyse, E. A., J. Exp. Med. 144, 1128, 1976. 6. Greene, M. I., and Benacerraf, B., Immunol. Rev. 150, 37, 1980. 7. Alter, B., and Bach, F. H., J. Immunol. 123, 2599, 1979. 8. Benacerraf, B., Behring Werk Mitt. 63, 56, 1979. 9. Black, H. S., and Lo, W. B., Nature (London) 234, 306, 1971. 10. Black, H. S., and Douglas, D. R., Cancer Res. 33, 2094, 1973. 11. Weissman, G., and Fell, H. B., J. Exp. Med. 116, 365, 1962. 12. Black, H. S., and Chan, J. T., Photobiology 26, 183, 1977. 13. De Rios, G. G., Rudolph, A. H., Chan, J. T., and Black, H. S., C/in. Res. 24, 263A, 1976. 14. Kripke, M. L., J. Nut. Cancer Inst. 53, 1333, 1974. 15. Prehn, R. T., and Main, J. M., J. Nat. Cancer Inst. 18, 769, 1957. 16. Old, L. J., Boyse, E. A., Clarke D., and Carssvell, E., Ann. N.Y. Acad. Sci. 101, 80, 1962. 17. Forbes, J. T., Nakao, Y.. and Smith, R. T., J. Exp. Med. 141, 1181, 1975. 18. Hellstrom, K. E., Hellstrom, I., and Brown, J. P., Int. J. Cancer 21, 317, 1978. 19. DeLuca, D., Kripke, M. L., and Marchalonis, J. J., J. Immunol. 123, 2696, 1979. 20. Kripke, M. L., and Fisher, M. S., J. Nat. Cuncer Inst. 57, 211, 1976. 21. Spellman, C. W., and Daynes, R. A., Transplantation 24, 120. 1977. 22. Kripke, M. L., Submitted for publication, 1979. 23. Daynes, R. A., Schmitt, M. K., Roberts, L. K., and Spellman, C. W., J. Immunol. 122, 2458, 1979. 24. Kripke, M. L., Cancer Res. 37, 1395, 1977. 25. Hsu, J., Forbes, P. D., Harber, L. C., and Lakow, E., Photochem. Photobiol. 21, 185, 1975. 26. Daynes, R. A., Spellman, C. W., Woodward, J. G., and Steward, D. A., Transplantation 23,343, 1977. 27. Norbury, K. C., Kripke, M. L., and Budmen, M. B., J. Nat. Cancer Inst. 59, 1231, 1977. 28. Fortner, G. W., and Kripke, M. L., J. fmmunol. 118, 1483, 1977. 29. Kripke, M. L., J. Reticuloendothel. Sot. 22, 217, 1977. 30. Fisher, M. S., and Kripke, M. L., Proc. Nat. Acad. ,‘j’ci. USA 74, 1688, 1977. 31. Howard, J. G., and Mitchison, N. A., Progr. Allergy 18, 43, 1975. 32. Gershon, R. K., and Kondo, K., Immunology 21, 903, 1971. 33. Fujimoto, S., Greene, M. I., and Sehon, A. H., J. Immunol. 116, 791, 1976. 34. Takei, F., Levy, J. G., and Kilburn, D. G., J. lmmunol. 118, 412, 1977.
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FOX ET AL. Kuperman, 0, Former, G. W., and Lucan, Z. J., J. Immunol. 115, 1282, 1975. Perry, L. L., Benacerraf, B., and Greene, M. I., J. Immunol. 121, 2144, 1978. Miller, S. D., Sy, M. S., and Claman, H. N., J. Immunol. 121, 274, 1978. Miller, S. D., Sy, M. S., and Claman, H. N., J. Immunol. 121, 265, 1978. Spellman, C. W., and Daynes, R. A., Cell. Immunol. 36, 383, 1978. Fujimoto, S., Greene, M. I., and Sehon, A. H., J. Immunol. 116, 800, 1976. Kontiainen, S., and Feldmann, M., Eur. J. Immunol. 6, 296, 1976. Taniguchi, M., and Miller, J. F. A. P., J. Exp. Med. 148, 373, 1978. Rosenthal, A. S., and Shevach, E. M., J. Exp. Med. 138, 1194, 1973. Perry, L. L., Kripke, M. L., Benacerraf, B., Dorf, M. E., and Greene, M. I., Cell. Immunol. 51, 349, 1980. Greene, M. I., Dorf, M. E., Pierres, M., and Benacerraf, B., Proc. Nat. Acad. Sci. USA 74, 5118, 1977. Parish, J. A., Anderson, R. R., Urbach, F., and Pitts, D. (Eds.), In “UVa-Biological Effects of UV Radiation with Emphasis on Human Responses to Long Wave UV” Plenum Press, New York, 1978. Lindahl-Kiessling, K., and Safwenberg, J., Int. Arch. Allergy 41, 670, 1971. Pertoft, H., Back, O., and Kindahl-Kiessling, K., Exp. Cell. Res. 50, 355, 1968. Lafferty, K. J., Nature (London) 249, 275, 1974. Setlow, R. B., and Carrier, W. L., J. Mol. Biol. 17, 237, 1966. Schellekens, P. M. A., and Einsvoogel, B. P., Clin. Exp. Immunol. 7, 229, 1970. Webb, R. B., and Lorenz, J. R., Photochem. Phofobiol. 12, 283, 1970. Peak, M. K., Photochem. Photobiol. 12, 1, 1970. Stoien, J. D., and Wang, R. J., Proc. Nat. Acad. Sci. USA 71, 3961, 1974. Wang, R. J., Stoien, J. D., and Landa, F., Nature (London) 247, 43, 1974. Wilson, D. B., J. Exp. Med. 126, 625, 1967. Amos, D. B., and Bach, F. H., J. Exp. Med. 128, 623, 1968. Sorensen, S. F., Acfa Pathol. Microbial. Stand. 78, 711, 1970. Majesky, A., Curr. Top. Microbial. Immunol. 50, 138, 1969. Hayry, P., and Anserssen, L. C., Stand. J. Immunol. 5, 391, 1976. Bach, F. H., Gullet-Courvalin, C., Juperman, 0. J., Sollinger, H. W., Hayes, C. Sondel. P. M., Alter, B. J., and Bach, M. L., Immunol. Rev. 35, 76, 1977. Bach, F. H., Bach, M. L., and Sondel, P. M., Nature (London) 259, 273, 1976. Rollinghoff, M., and Wagner, H., Eur. J. Immunol. 5, 875, 1975. Levis, W. R., Lincoln, P. M., and Dattner, A. M., J. Immunol. 121, 1496, 1978. Alter, B. J., Schendel, D. J., Bach, M. L., Bach, F. H., Klein, J., and Stimpfling, J., J. Exp. Med. 137, 1303, 1973. Schendel, D. J., and Bach, F. H., In “Lymphocyte Recognition and Effector Mechanisms” (K. Lindahl-Kiessling and D. Osaha, Eds.), Academic Press, New York, 1974. Kasakura, S., J. Immunol. 105, 1162, 1970. Lawrence, H. S., and Landy, M. (Eds.), In “Mediators of Cellular Immunity,” Academic Press, New York, 1969. Lafferty, J. K., Walker, K. Z., Scollay, R., and Killby, V. A., Transplant. Rev. 13, 198, 1972. McFarland, W., and Hellman, D., Nafure (London) 205, 887, 1965. Corley, R. B., Dawson, J. R., and Amos, D. P., Cell. Immunol. 16, 92, 1975. Lafferty, K. J., Misko, I. S., and Cooley, M. A., Nature (London) 249, 275, 1974. Engers, H. D., Thomas, K., Cerottini, J. C., and Brunner, K. T., J. Immunol. 115, 356, 1975. Levis, W. R., and Robbins, J. H., Transplant. Bull. 9, 515, 1970. Rosenthal, A. S., and Shevach, E. M., Curr. Top. Immunobiol. 5, 47, 1976. Levis, W. R., and Robbins, J. H., J. Immunol. 104, 1295, 1970. Levis, W. R., and Robbins, J. H., Blood 40, 77, 1972. Manson, L. A., and Simmons, T., Transplant. Proc. 1, 498, 1969. Wagner, H., and Boyle, W., Nature New Biol. 240, 92, 1972. Wagner, H., Hess, M., Feldman, M., and Rollinghoff, M., Transplantation 21, 282, 1976. Levis, W. R., Whalen, J. J., and Sherins, R. J., Lancer 2, 954, 1974.
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Levis, W. R., Whalen, J. J., and Sherins, R. J., Science 191, 302, 1976. Halim, K., and Festenstein, H., Lancer 2, 1255, 1975. Levis, W. R., and Dattner, A. M., J. Immunol. 122, 1986, 1979. Edwards, R. G., Proc. Roy. Sot. London 146, 488. 1957. Trotter, T. M., and Armstrong, J. B., Genetics 83, 783, 1976. Haniszko, J., and Suskind, R. R., J. Invest. Drrmatol. 40, 183, 1963. Greene, M. 1.. Sy. M-S., Kripke, M. L., and Benacerraf, B., Proc. Nat. Acad. SC;. USA 76,6591, 1979. 89. Toews, G. B., Bergstresser, P. R., and Streilein, J. W., J. Immunol. 124, 445, 1980.
IMMUNOLOGY
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IMMUNOPATHOLOGY
155 (1980)
REVIEW The Influence
IRA J. Fox, LINDA
of Ultraviolet Light Irradiation Immune System’ L. PERRY, MAN-SUN
SY, BARUJ BENACERRAF, I. GREENE
MARK Department
on the
oj’ Pathology, Harvard Medical Boston, Massachusetts Received
March
School, 02115
25 Shattuck
AND
Street,
13, 1980
INTRODUCTION
Ultraviolet (uv) light irradiation has been associated with the development of sarcomas and carcinomas (1). This phenomenon has been best studied in mice, but similar effects occur in humans (2). Recently, the “escape” of uv-induced tumors has been shown to be related to the influence of ultraviolet light on the immune system (1, 3). The immune response to antigens reflects the participation of many elements of the immune system. Thymus-derived (T), bone marrow-derived (B), and antigenpresenting (APC) cells (probably of monocytic origin) are required for many humoral responses (4). Furthermore, cellular reactivity of T cells reflects the interactions of T-cell subsets defined by distinct differentiation antigens of the Lyt series (5). Thus, Lyt 1+23- T helper cells can augment target cell lytic responses mediated by Lyt l-23+ T cytotoxic cells (6). Delayed-type hypersensitivity (DTH) reactivity is mediated by Lyt l+ T cells, although synergistic interactions with other T-cell subsets such as Lyt 123+ cells may also be required for maximal responsiveness (6,7). Cells which proliferate in vitro in response to antigen-pulsed APC are Lyt l+ cells. The interaction and clonal expansion of these T-cell subsets after exposure to antigen is subject to regulation by T suppressor cells (Ts). Ts function is complex and in some experimental systems is dependent upon at least two interacting subsets of suppressor cells (8). Antigen activates first-order Ts termed Ts, which release soluble suppressor factors (TsF) that induce secondorder T cells (Ts,) to limit immune reactivity (8). In this review, we shall provide evidence that a critical cellular component necessary for immune responses is uniquely impaired by uv irradiation. It has also been possible, employing uv treatment, to elucidate multiple levels of antigen recognition which could serve as targets for immune regulation. We will then consider the possible immunological mechanisms involved in uv-induced tumor enhancement and discuss how the use of uv irradiation might provide a practical approach to graft rejection. ’ Supported
by Grants
CA-14732
and AI-16396. 141 0090-1229/80/090141-15$01.00/O Copyright All rights
0 1980 by Acadermc Press, Inc. of reproduction m any form reserved
142
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ET AL.
IRRADIATION
OF CARCINOGENESIS
Tumor induction as a consequence of ultraviolet (uv) light irradiation has been studied in humans and in experimental animal models (1, 2). Photobiologists have found that uv-induced tumor development reflects a variety of cellular alterations associated with treatment with particular spectra of uv light. These may include the photochemical conversion of cell-bound steroids into carcinogenic compounds (9, lo), the release of liposomal hydolases as stimulators of inflammation and damage to essential cellular proteins (11, 12), and the cross-linking and dimerization of host cell DNA (reviewed in (12)). The protective capacity of ingested antioxidants has suggested a role for dietary factors in uv carcinogenesis as well (12, 13). With the possible exception of damage and/or faulty repair of uv-treated DNA, experimental evidence to support these proposed mechanisms of photocarcinogenesis is lacking, however (12). More recently, the relationship between uv light treatment and the expression of host cell-mediated immunity has been investigated. These productive studies have shed some light on the cellular basis of susceptibility to tumor growth in uv-treated animals. Tumors induced in mice by prolonged exposure to uv light irradiation display a high degree of antigenicity. Such tumors are indeed frequently rejected upon transplant to normal syngeneic recipients (14) (see Fig. 1). The antigens responsible for tumor rejection appear to be largely tumor specific, since normal animals immunized against a particular uv-induced fibrosarcoma and then subjected to sublethal X irradiation are capable of rejecting only the immunizing tumor upon subsequent rechallenge (14). The antigenic properties of uv tumors are similar to those of chemically induced neoplasms, in that tumors developing as a result of treatment with agents such as methylcholanthrene (MCA) generally display unique tumor-specific transplantation antigens (TSTA) even when tumors arise in the same inbred strain or host (15, 16). There is increasing evidence to indicate, however, that the antigens expressed by MCA-induced tumors may contain common or cross-reactive determinants as well as unique TSTA. In vitro (17) proliferation assays and in vivo transplantation studies (18) have demonstrated common antigens among tumors. The presence of cross-reactive antigens has also been suggested in the case of uv-induced tumors, by serological analysis (19), and by tumor transplantation. Thus, mice hyperimmunized against a particular uvinduced fibrosarcoma by multiple injections of tumor cells may reject other uvinduced tumors to which they did not previously respond. Precise definition of the determinants contributing to tumor resistance in the uv system is lacking, therefore, and may involve a variety of antigenic target structures. Resistance against uv-induced tumor transplants appears to be mediated primarily by cellular rather than humoral immune mechanisms. For example, animals which have been rendered immunodeficient by adult thymectomy and X irradiation (20) or by treatment with cyclophosphamide (21) or anti-lymphocyte serum (21) demonstrate a reduced in vivo response against the tumor. Normal responsiveness can be restored to such animals by the adoptive transfer of lymphocytes from normal or tumor-immune donors (14, 22). The subsets of cells responsible for the protection have been recently shown to be T cells by Daynes and colleagues (23).
ULTRAVIOLET
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ON
. 2~10~1316 I” Normal C3H 0 2x to6 1316 in UV Treated C3H . 2 x i06 I316 in Immune UV Treated
THE
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Mice
DAYS
FIG. 1. uv-Induced tumor growth in uv-treated, normal or immune uv-treated mice. C3H uv-treated mice were prepared for use by Dr. M. Kripke and were challenged with 2 x 10” 1316 fibrosarcoma (tumor also supplied by Dr. Kripke). Seven days later the tumor was removed and 28 days later these mice were rechallenged with the relevant tumor. Normal C3H, or uv-treated C3H, were also challenged at that time with 2 x lob: 1316. Tumor growth was followed daily. Whereas normal or immune uv-treated mice rejected tumor, uv-treated mice did not. It should be noted, however, that many weeks (>6) had elapsed between the time the uv-immune group were last treated with uv irradiation and the experiment depicted above.
In contrast to the spontaneous rejection response of normal syngeneic hosts, animals previously treated with subcarcinogenic levels of uv irradiation demonstrate progressive tumor growth when challenged with uv-induced antigenic tumor cells (20). The dosage of irradiation necessary to induce tumors varies between individual tumor lines and inbred host strains; nude or albino mice requiring less exposure than agouti or black animals (24). In certain cases, this effect can be accomplished with a single prolonged uv light treatment (26), suggesting that total dose rather than duration of treatment may be the determining factor (26). Definition of the cellular basis of the defective anti-tumor response in the uv-treated host has been approached by examining multiple parameters of T-cell and macrophage activation, in vivo as well as in vitro. Results have shown that although uv-treated mice are incapable of rejecting syngeneic uv-induced tumor grafts, their response against H-2 incompatible tumor allografts and H-2 allogeneic or semisyngeneic skin grafts appears normal (20). Other assays of immune function such as primary hemagglutinin responses to sheep erythrocytes and inflammatory reactions against dimethyl sulfoxide are also equivalent to those of untreated hosts (1,3). Analysis of the functional nonspecific reactivity in vitro of lymphoid cells from uv-treated mice has revealed normal patterns of responsiveness to concanavalin A and lipopolysaccharide and equivalent levels of macrophage tumoricidal and phagocytic capabilities (27). In vitro cytotoxicity against allogeneic or hapten-modified syngeneic cells (3) was also uninhibited, while, under conditions of a 48-hr microcytotoxicity assay, little or no cytotoxic activity
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was apparent against uv-induced tumor cells (28). The defect induced by uv treatment appears to be detectable only under specific conditions. Indeed, generally, the uv-treated host does not display an accelerated tumor growth rate when challenged with tumors induced by chemical carcinogens or other unrelated means (29). On the basis of these findings, it was suggested that uv treatment may induce a state of specific nonresponsiveness or tolerance to antigens expressed on uvinduced tumors (21, 30). Tolerance to an antigen has been shown to occur by at least two mechanisms, one involving the inactivation of T-cell clones specific for a given antigen by either clonal abortion, receptor blockade, or other ill-defined means (31), and another reflecting the generation of suppressor T cells (Ts) that have the capability to inhibit the expression of specific T-cell effector responses (32). Suppressor T cells have been shown to influence immunity to a variety of tumor types (33-35), such that Ts which arise during primary tumor development suppress the expression of T-cell immunity to tumor antigens, and, as a result, inhibit tumor rejection (33). Suppressor T cells in many systems are characterized by the expression of antigens encoded by the I-J subregion of the murine histocompatibility complex (MHC), and by the elaboration of I-J+ factors that participate in a suppressor cell circuit to induce secondary antigen-specific Ts (36). Depending on the system employed, Ts may suppress either the afferent (37) or the efferent (38) limb of a cellular immune response. Investigations into the possible contribution of suppressor cells in the enhancement of uv-induced tumors by previous uv irradiation revealed that these cells may also play a role in the proposed tolerance to uv tumor antigens (21, 30, 39). This has been demonstrated by a variety of means. First, it was found that tumor immunity transferrable to lethally X-irradiated recipients by normal syngeneic cells could be abrogated by the cotransfer of lymphoid cells from uv-irradiated mice (22,30). Second, susceptibility to uv-induced tumor growth could be conferred onto normal individuals by joining the latter in parabiosis with uv-treated animals (30). Finally, the transfer of spleen cells from uv-treated mice into normal recipients inhibited the normal animal’s capacity for rejection of uv-induced tumors (21). The cells responsible for this suppression were shown to be derived from the thymus (21) and sensitive to the effects of anti-Thy 1 antiserum plus complement (21), in accordance with the phenotype of Ts operative in other tumor systems (40). Interestingly, the presence of a growing tumor in uv-irradiated animals is not required for the generation of suppressor cell activity (21, 30). This observation raises some interesting and important questions regarding the mechanism of suppressor cell generation and the specificity of the reactive cell. According to current theories, Ts are stimulated by the presence of excess free antigen, such as that shed from the surface of live or disintegrated tumor cells (41). This hypothesis derived from the observations that Ts can be generated by antigen in macrophage-free cultures in vitro (41) and can bind to antigen-coated plates (42), indicating specificity for antigen alone. In the case of uv-induced Ts, it could be argued that cellular alterations that may accompany uv treatment, as the dimerization and faulty repair of host cell DNA, may result in the creation of new antigenic structures on the surface of affected cells similar to those expressed by uv-induced tumors. The steps leading from these changes to the stimulation of Ts
ULTRAVIOLET 100-
Bop
INFLUENCE
ON THE IMMUNE
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145
UV lrradloied C3H o 2x 10~ 1316 (H-29 + 21NMS/day .2 x106 1316 + Zl/day (~6~12 x BI~A (3R1) F, Ant!-BlOA (5R)
FIG. 2. uv-Treated C3H or normal C3H mice were challenged with 2 x 10” 1316 (H-2”) cells subcutaneously, on Day 0. Beginning at the time of tumor challenge, selected groups also received 2 PI/day/mouse of normal serum of 2 ~1 of anti-I-Jk antiserum (DBA12 X BIO.A(3R))F, anti-BlO.AtSR). Anti-I-Jk-treated mice had highly significantly reduced tumor growth.
could then be the result of failure to present antigen in the appropriate context of macrophage Ia determinants, which is normally required for the activation of positive effector T-cell activity (43). Evidence for the dysfunction of antigenpresenting cells in the uv-treated host will be presented in the following sections of this review. Transplantation of uv-induced tumors into the uv-treated host may stimulate additional suppressor cell activity, with specificity for antigens of the uv tumor. That these Ts contribute to progressive tumor growth in the uv host has been demonstrated after eliminating suppressor activity in viva. This has been accomplished by treatment of the uv-treated tumor-bearing host with alloantiserum specific for I-J subregion encoded determinants (44) using a protocol that has been shown to inhibit effectively Ts generated by chemically induced tumors (45) (see Fig. 2). Under these conditions, tumor growth in the antibody-treated host is significantly reduced and in some cases completely halted (44). Therefore, the failure of the uv-treated host to eliminate a tumor challenge successfully is probably (i) the result of the activation of multiple clones of antigen-specific Ts, including those induced by the tumor as well as those activated by the uv treatment itself, and (ii) the consequence of shared specificities for uv-induced antigens and uv-induced tumor cells. The underlying basis for Ts stimulation and for the apparently normal responses to mitogens and allogeneic stimuli will be elucidated further below. IN VITRO ANALYSIS OF THE CELLULAR EFFECTS OF ULTRAVIOLET IRRADIATION ON THE IMMUNE SYSTEM
The data strongly suggest that there exists a specific defect in immunity arising from irradiation with uv light and resulting in the stimulation of specific Ts. The
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precise mechanism by which this alteration occurs, however, has remained obscure. Several investigators have suggested that experiments using in viva models involve multiple, complicated mechanisms, all of which may not be controlled for within an experiment. In order to avoid the pitfalls associated with such work, these researchers have chosen to study the effects of uv light directly on cells involved in the immune system by in vitro analysis. In this regard, it has been found that lymphocytes are easily inactivated by ultraviolet light; their viability is compromised in vitro by as little as 100 mJ/cm2 (46). At uv doses which do not influence viability as judged by trypan blue exclusion (47, 48), these uv-treated lymphocytes nevertheless demonstrate little DNA synthesis in response to mitogens such as phytohemagglutinin (PHA) (47). Membrane properties such as the expression of alloantigen (determined by H-2 antibody absorption and cytotoxicity with complement) and the binding of PHA are apparently uninfluenced by uv irradiation (47, 49). In addition, irradiated cells do not appear to have acquired toxic properties. For example, it is well established that both free PHA and PHA bound to syngeneic lymphocytes stimulate responding cells to proliferate. It has been shown that uv-irradiated lymphocytes bearing PHA are equally efficient inducers in this system. It is also well established that if either prokaryotic or eukaryotic cells are irradiated directly with uv light, DNA replication is inhibited by the formation of pyrimidine dimers (50). Similar inhibition has been reported with respect to RNA and protein synthesis (46). Further, ultraviolet light treatment has been reported to cause induction, inactivation, or alteration in enzyme function (51). It is now known that these changes may reflect more than just the direct mutagenic effect of uv irradiation. Such changes in gene expression may, for example, be a cellular response to generalized stress induced by ultraviolet irradiation. Since different uv wavelengths elicit different biological effects, there is some confusion as to the nature of the effect which causes altered lymphocyte function; alterations generated by irradiation in the A region (320-400 nm) of uv, for example, appear to be oxygen dependent in certain systems, whereas C region (200-290 nm) uv light-induced damage is relatively oxygen independent (52, 53). Hence, the mechanism of A region-induced change must be different from that of C region-induced change. In addition, it has been demonstrated that uv irradiation of tissue culture medium containing cell nutrients can result in the death of cells growing in that medium (51, 54,55). Presumably, this is a result of the generation of toxic by-products. The consequences of such an effect on the study of immunocompetent cells are important since the presence of photoproducts in media may lead to misinterpretation of the effects of uv light on in vitro systems. Irradiation of cells in nonnutritive medium should eliminate such effects and exclude this potential artifact. THE INFLUENCE
OF ULTRAVIOLET
LIGHT ON ALLOREACTIVITY
It has been generally accepted that a difference in certain major histocompatibility complex (MHC)-encoded antigens on the cell membrane between a stimulator and a responder cell is critical for inducing proliferation in the mixed lymphocyte reaction (MLR) (56-58). To what extent these MHC-encoded anti-
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SYSTEM
147
gens by themselves determine immunogenicity has never been fully defined. For example, A strain platelets, though carrying some of the same histocompatibility antigens as A strain lymphocytes, are incapable of inducing the same degree of MLR stimulation to B strain allogeneic cells (59). This observation raises doubt as to whether MHC antigen recognition alone accounts for MHC immune responsiveness. It has been suggested that only adherent cells are capable of acting as potent stimuli for the MLR in vitro. T cells or B cells, in the absence of adherent cells, provide little MLR stimulus to T-cell responders. It may be that allogeneic determinants might be presented to responder cells by antigen-presenting cells within the adherent cell population. Irradiation of immunocompetent allogeneic cells with uv light has been used to help determine which signals mediate antigenic-specific T-lymphocyte activation. Work in several laboratories has convincingly demonstrated that purified splenic T lymphocytes are unable to respond to uv-irradiated allogeneic cells in primary mixed lymphocyte and cytotoxicity assays (47, 51, 60, 61) (Fig. 3). In contrast, following in vitro mixed lymphocyte sensitization with normal cells, “secondary’‘-type mixed lymphocyte and cytotoxicity responses have been elicited by restimulation with uv-irradiated cells in both mouse and human systems (49, 60-64). However, if responding cells, in a primary stimulation, are confronted simultaneously with an ultraviolet-irradiated cell and a second cell differing only at the I region with the responding cell, a strong cytotoxic response to the K or D region of the uv-irradiated cell is elicited (61, 65, 66). Therefore, uv treatment seems to abrogate I region-associated stimuli required for activation of such immune cells. Although I region products appear to be affected, the specific defect induced by irradiation with ultraviolet light is at present unclear. There are several possible targets. Several investigators have demonstrated biologically potent growth factors, T-cell stimulation factors, and other products which are elaborated from cells of the immune system (67-69). The production of these products by either macrophages and/or lymphocytes may well be sensitive to uv I
I - 60,000
Stimulators
l 5xlO6/ml 0 1 x 106/ml
- 50,000 - 40,000
A 5 x 10s/ml
- 30,000 - 20,000
8 b P
- I0,000 -0 I1 0
1
IO T/ME
I
I
20 EXPOSED
II
I
30
I
40
I
I
50
TO UV LIGHT
I
I
60
I
fsecl
FIG. 3. The ability of uv light to interfere with MLR. A/J (H-29 mouse spleen cells were used as responders to mitomycin C-treated BALBic (H-2”) stimulator cells that had been exposed to uv light for the time periods indicated on the abscissa. The stimulator cells were splenic adherent cells that had been recovered from two cycles of adherence and separation from plastic plates. The uv light was delivered using a bank of FS40 sun lamp lights (2.0 J/m”/sec of 280-340-nm light).
148
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ET AL.
light. Alternatively, since antigenically stimulated cells make contact with other cells in culture by membrane extensions (70), uv-treated cells may have simply lost some aspect of their motility and, therefore, present antigen poorly. uv Irradiation may modify cell surface antigens and membranes, causing a loss or alteration of relevant molecules needed for immune responses. Indeed, following sensitization in viva with uv light-treated allogeneic lymphocytes, one can obtain very strong in vitro cytotoxicity responses to uv-treated cells (62). However, mice primed to untreated allogeneic cells respond in a limited manner to uv-treated allogeneic stimulators in vitro. The argument could be raised that by irradiating lymphocytes with uv light one induced a structural modification in the histocompatibility antigens. It is possible to propose a mechanism for T-cell activation from the above data. Bach and others have proposed a model to account for T-cell activation requiring two discrete signals (62). Signal 1 would be mediated by the antigenic target, which on the mouse cell would be either K or D region determinants. Signal 2, which is sensitive to uv irradiation and derived from I region-associated products, would, on the other hand, stimulate a separate population of proliferating helper cells which are necessary to maximize the response to K and D targets. This model should be extended to include antigen-presenting cells (APC), which are an integral part of T-cell activation. In this regard, it has been shown that membrane extracts and subcellular preparations will stimulate resting allogeneic lymphocytes in vitro if and only if a non-T-cell population is present in the responding population, presumably to present antigen (69, 71-80). Indeed, there is some evidence that the population of APC can be supplied by either the stimulating or responding population in alloreaction (74). It is likely, as will be discussed later, that the APC is the target of uv irradiation. There are, however, several inconsistencies in the literature with regard to work involving uv light. It has been reported that human sperm cells are not only capable of stimulating allogeneic lymphocytes, but that these cells are resistant to uv light when inducing primary allogeneic proliferative responses (81-84). Interestingly, sperm cell antigens cross-react with histocompatibility determinants on leukocytes but cannot restimulate their own primary sensitization, In vitro sensitization with sperm cells results in alloantigen-specific secondary MLR when leukocytes are used for restimulation but not when sperm cells are used for restimulation (84). Whether T-cell suppression of the sperm cell-induced immune reaction occurs when sperm cells are used to restimulate in the latter case is not known. However, there remains some doubt as to the general applicability of analyzing cell surface antigens of sperm to study alloreactivity or the effects of uv on the immune system. For example, it is well established that the inactivation of sperm DNA requires a higher dose of uv irradiation than does DNA from other tissues (85, 86). It should be noted as well that uv-induced changes in secondary MLR are also variable. Bach et al. could show that secondary responses are only inconsistently triggered by uv-treated cells, whereas others have found consistent restimulation (61). The explanation for these differences is not clear. It is known, however, that leukocytes treated with uv light and held overnight are much less efficient in hapten-presenting function to primed cells than leukocytes that are
ULTRAVIOLET
INFLUENCE
ON
THE
IMMUNE
SYSTEM
149
irradiated and added to culture within a few hours (84). Insufficient control of such factors may lead investigators to erroneous conclusion, and one can only postulate other as yet undefined variables associated with uv effects on cells. Whatever the mechanism, it is clear that some function (probably involving I-region-bearing cells, such as APCs) is sensitive to uv irradiation. This function is an essential component of the immune response, since, once ablated, effector immune reactions are not induced. In terms of recognizable effects on animals treated with uv irradiation, aside from the aforementioned tumor model, other work has consistently demonstrated that uv-treated animals possess little or no generalized alteration in immune function (27). Indeed, such animals, as previously mentioned, though unable to reject uv-induced syngeneic tumors, rejected both H-Zincompatible skin and tumor allografts (1). In addition to those studies described above, lymphocytes from animals receiving chronic ultraviolet irradiation in doses capable of inducing primary tumors maintained (i) normal plaque-forming cell response to PVP, (ii) normal primary cytotoxicity in vitro to non-uv-induced syngeneic tumors, and (iii) normal induction of local GVH reaction (normal animal recipients of lymphocytes from uv-irradiated donors) ( 1- 3). However, some alteration in the immune response of these animals has been demonstrated. Lymphocytes from irradiated animals display a 20-50% lower proliferative response to MLC stimulation than do lymphocytes from untreated animals (3). Similarly, uv-irradiated animals developed a transiently depressed GVH response to normal allogeneic cells during the early months of irradiation. In addition, delayed hypersensitivity to dinitrochlorobenzene (DNCB) was also found to be transiently depressed but returned to normal prior to the development of primary tumors (1, 5). Since the above changes in immunity were present only early during treatment and did not correspond to the development of tumors, there was some concern whether these mechanisms were operating in uv carcinogenesis. In vitro work was initiated to look for altered macrophage function since two of the alterations noted above primarily measured macrophage infiltration. Macrophages from uv-treated mice displayed (i) normal phagocytosis following opsinization, (ii) normal PEC induction in response to inflammatory agents, and (iii) normal in vitro tumoricidal capacity (27). In addition, spleens from irradiated animals contain a normal distribution of &bearing, immunoglobulin-bearing, and adherent cells when compared with spleens from normal animals. Thus, these studies were unsuccessful at localizing a defect. It was only when detailed in vivo studies using exogenous antigens were performed that a specific immunologic defect was identified that may explain, at least in part, uv-induced carcinogenesis. EFFECT
OF ULTRAVIOLET IRRADIATION HYPERSENSITIVITY
The first report ability to mount made by Haniszko of guinea pig skin
ON DELAYED-TYPE
indicating that uv-irradiated animals may be defective in their effective delayed-type hypersensitivity (DTH) responses was and Suskind (87). These investigators found that uv irradiation results in unresponsiveness to topical challenge with a contac-
150
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ET
AL.
tant, dinitrochlorobenzene (DNCB). However, if challenge was done at an unirradiated site on the same animal, the contactant elicited an apparently normal DTH response. Thus, the effect of limited uv-irradiation in the guinea pig appeared to be localized only in the exposed area. More recently, extensive studies on the effect of uv irradiation on DTH immunity in the murine system have been performed in the laboratory of Kripke and colleagues (1, 5, 14). Consistent with the above-mentioned, earlier findings, these investigators found that uv-treated mice displayed a transient unresponsiveness in DNCB contact sensitivity. This alteration lasted for the first 2 to 3 months of uv irradiation, after which these animals recovered normal responsiveness. The mechanisms by which uv irradiation brought about this transient reduction in DTH was not clear, as lymphocytes from uv-treated DNCB-primed mice could transfer immunity to naive recipients. Therefore, it was postulated that the de& ciency might reside in the earlier antigen-processing steps of the immune response . Definitive evidence of an antigen-presenting cell defect in uv-irradiated mice was provided in recent experiments by Greene et al. (88). These studies were made possible when it was found that a state of hapten-specific DTH could be induced in normal mice by the subcutaneous injection of hapten-conjugated syngeneic spleen cells. Fractionation of spleen cells according to their adherent properties on plastic petri dishes revealed that haptenated adherent cells were the most potent inducers of DTH as compared to similarly treated nonadherent cells. To examine the effect of uv irradiation on antigen-presenting cell function, normal and uv-treated BALB/c mice were immunized with TNP-conjugated normal adherent cells or TNP-conjugated adherent cells from uv-treated animals. The results, shown in Table 1, clearly demonstrate that normal animals develop significant DTH irrespective of the source of adherent cells. In contrast, uv-treated animals failed to produce significant levels of immunity when they were immunized with TNP-adherent cells obtained from uv-treated animals. This defect could be circumvented by immunization with TNP-derivatized adherent cells from normal mice, stimulating significant levels of DTH compatible to that of normal mice. From the experiments (depicted in Table 1, Experiment I), it was concluded that adherent cells from mice irradiated with uv light had lost their antigenpresenting cell function, and that the ability of TNP-conjugated uv-adherent cells to induce significant immunity in normal hosts was probably due to representation of antigen by the host antigen presenting cell. Most important was the finding (Table 1, Experiment II) that immunization of uv-treated mice with haptenderivatized cells obtained from uv-treated donors led to the generation of haptenspecific suppressor T cells. These Ts, upon adoptive transfer to normal recipients, abrogated their ability to respond to hapten-conjugated cells. Thus, uv light not only interferes with APC function but, as a consequence, predisposes the animal to the generation of Ts to any antigen which the APC would normally present. DISCUSSION
It has been difficult to determine whether uv irradiation directly alters host immunity. Multiple in vitro and in vivo assays of B-cell, T-cell, and phagocyte
uv uv -
IV V VI VII
TABLE
I
IO’ uv Tnp spl adh IO’ uv Tnp spl adh -
10’ uv Tnp spl adh IO’ uv Tnp spl adh
-
lo7 Tnp spl adh IO’ uv Tnp spl adh -
-
Immunizing cell”
-
5 X 10’ spleen cells
-
5 x IO7 spleen cells 5 x 10’ spleen cells (anti-Thy I.2 + C treated) 5 x IO’ spleen cells (NMS + C’)
-
-
5 X IO’ spleen cells 5 X IO7 spleen cells
-
Cells transferred to second recipient
7%, TNCB 0.5% DNFB 0.5% DNFB -
7% TNCB 7% TNCB 7% TNCB
7% TNCB 7% TNCB 7% TNCB -
1% TNCB 1% TNCB 1% TNCB
1% TNCB 1% TNCB
1%TNCB
1% TNCB
Challenge second recipient” -t k 5 k
5.0 4.0 4.0 3.0
26.0 85.0 86.0 6.0
‘f + -c
1.0 5.0 5.0 5.0
47.0 ? 5.0 27.0 -t 2.0 42.0 k 5.0
71.0 72.5 37.0 18.0
lo-” in ? SEM
APC LEADS TO THE
1% TNCB 0.2% DNFB 0.2% DNFB 0.2% DNFB
DERIVED
Immunization of second recipienY
WITH HAPTEN-DERIVATIZED W-TREATED APC BUT NOT NORMAL GENERATION OF SUPPRESSOR T CELLS
ns
-
P’
” Ultraviolet light irradiation of shaved mice for 1 hr. three times a week for I month using the FS40 sun lamp (2.0 J/m’/sec of 280-340-nm light). ” Adherent (spl adh) or nonadherent cells from the spleen (6) or peritoneal exudate cells (PEC) were derived with 10 m&f TNBS as described (688). ’ Immunization with 7% PCI (6) or 0.5% DNFB on the shaved abdomen as described previously (I). ” Challenge was performed using 1% TNCB in olive oil on the ear on Day 5. Measurements using an engineer’s caliper were performed 24 hr later. The increment of the challenged ear above the unchallenged was measured and recorded. (’ P values determined by the two-tailed Student’s I test.
uv
uv
uv -
uv
Expt II 1 II 111
Expt I I 11 III 1V
Group
Treatment of mice”
IMMUNIZATION
152
FOX
ET AL.
function appear to be unaffected by uv light (1, 3, 14, 20-22). Several studies investigating antigen presentation to T cells in viva, however, suggest that uv irradiation does alter cellular immunity. Research in contact sensitivity and delayed-type hypersensitivity has confirmed such suspicions. In the systems under study, uv irradiation has resulted in a depressed DTH response, which is mediated by antigen-specific Ts. uv-Irradiated animals maintain normal T- and B-cell function, but are capable of mounting a normal immune response only if antigen is appropriately presented, for example, as seen in Table 1, when splenic adherent cells covalently coupled with hapten obtained from a normal animal are used for presentation. Splenic adherent cells obtained from uv-irradiated animals and then coupled with hapten are unable to induce a normal CS response in uv-irradiated hosts. Therefore, some aspect of antigen presentation is altered in these animals. Since cells within the spleen display this alteration, the ability to present antigen must be the function of a pool of cells which can be affected by nonpenetrating uv skin irradiation. It is likely that APCs function by recirculating between central and peripheral lymphoid compartments where they routinely encounter foreign antigens. uv Irradiation has been shown to decrease the number of I-A+ cells present at the site of irradiation. Therefore, if irradiating mice with uv light kills or alters APCs directly, the normal trafftc of such cells through the skin would eventually create an environment where APCs, once destroyed, could not be replaced. The mechanisms involved in Ts induction are not as yet delineated and are complex. If antigen is coupled to splenic adherent cells, and these cells are then injected iv into normal recipients, such animals generate antigen-specific Ts (37, 38). In at least one system, it appears that the ability to generate suppression is dependent upon APC presentation of antigen (44). However, in uv-irradiated animals such Ts seem to be generated as a consequence of impaired APC function. This mechanism may account for uv-induced photocarcinogenesis. Cellular alterations associated with uv treatment may result in the formation of new antigenic structures on the surface of affected cells; uv-induced tumors may develop tumor-specific antigens in just this manner. Since these antigenic structures are presented to the immune system by uv-impaired APCs, mechanisms similar to those found in contact sensitivity probably induce I-J+ Ts which are specific for the uv-induced neoantigens (44). Future work using the effects of uv light should help determine whether some special APC is needed for Ts activation. There is preliminary evidence indicating that the Langerhans cell, a type of monocyte found in the epidermis, is critical for inducing immunity in contact sensitivity. Investigators have noted a simple relationship between the density of Langerhans cells at the site of priming and the generation of immunity or suppression (89). Whether this implies that functionally different APC subsets modulate immune responsiveness is not yet known. The developmental maturity of APCs might, for example, play a pivotal role in the issue of immunity versus tolerance; or alternatively, entirely distinct APC cells may be responsible for these diverse effects. Direct uv irradiation of immunocompetent cells has also demonstrated that
ULTRAVIOLET
INFLUENCE
ON
THE
IMMUNE
SYSTEM
153
antigen recognition is mediated by at least two signals which result in immunity. One signal has been shown to be uv sensitive and is associated with cells bearing I-region gene products. Whether uv irradiation alters Ia surface antigens or affects the APC cell itself has not been determined. Work is presently under way to define what aspect of I-region-associated function is altered. In a similar manner, the uv system may help delineate the signals which function in the suppressor pathway. It is hoped that future work using uv light will continue to provide insight into the issue of how antigen processing relates to the generation of suppression and self-recognition. It may be possible to render grafts less susceptible to rejection, perhaps by uv irradiation of the recipient as well as the graft. Such treatment might result in the development of Ts specific for the relevant foreign antigens. At present experiments of this nature are underway in our laboratory. REFERENCES 1. Kripke, M. L., Lofgreen, J. S., Beard, J., Jessup, J. M., and Fisher, M. S., J. Nat. Cancer Inst. 59, 1227, 1977. 2. Marshall, V. C., Transplantation 17, 272, 1974. 3. Spellman, C. W., Woodward, J. G., and Daynes, R. A., Transplantation 24, 112, 1977. 4. Katz, D. H., and Benacerraf, B., Transplant. Rev. 22, 175, 1975. 5. Huber, B., Cantor, H., Shen, F. W., and Boyse, E. A., J. Exp. Med. 144, 1128, 1976. 6. Greene, M. I., and Benacerraf, B., Immunol. Rev. 150, 37, 1980. 7. Alter, B., and Bach, F. H., J. Immunol. 123, 2599, 1979. 8. Benacerraf, B., Behring Werk Mitt. 63, 56, 1979. 9. Black, H. S., and Lo, W. B., Nature (London) 234, 306, 1971. 10. Black, H. S., and Douglas, D. R., Cancer Res. 33, 2094, 1973. 11. Weissman, G., and Fell, H. B., J. Exp. Med. 116, 365, 1962. 12. Black, H. S., and Chan, J. T., Photobiology 26, 183, 1977. 13. De Rios, G. G., Rudolph, A. H., Chan, J. T., and Black, H. S., C/in. Res. 24, 263A, 1976. 14. Kripke, M. L., J. Nut. Cancer Inst. 53, 1333, 1974. 15. Prehn, R. T., and Main, J. M., J. Nat. Cancer Inst. 18, 769, 1957. 16. Old, L. J., Boyse, E. A., Clarke D., and Carssvell, E., Ann. N.Y. Acad. Sci. 101, 80, 1962. 17. Forbes, J. T., Nakao, Y.. and Smith, R. T., J. Exp. Med. 141, 1181, 1975. 18. Hellstrom, K. E., Hellstrom, I., and Brown, J. P., Int. J. Cancer 21, 317, 1978. 19. DeLuca, D., Kripke, M. L., and Marchalonis, J. J., J. Immunol. 123, 2696, 1979. 20. Kripke, M. L., and Fisher, M. S., J. Nat. Cuncer Inst. 57, 211, 1976. 21. Spellman, C. W., and Daynes, R. A., Transplantation 24, 120. 1977. 22. Kripke, M. L., Submitted for publication, 1979. 23. Daynes, R. A., Schmitt, M. K., Roberts, L. K., and Spellman, C. W., J. Immunol. 122, 2458, 1979. 24. Kripke, M. L., Cancer Res. 37, 1395, 1977. 25. Hsu, J., Forbes, P. D., Harber, L. C., and Lakow, E., Photochem. Photobiol. 21, 185, 1975. 26. Daynes, R. A., Spellman, C. W., Woodward, J. G., and Steward, D. A., Transplantation 23,343, 1977. 27. Norbury, K. C., Kripke, M. L., and Budmen, M. B., J. Nat. Cancer Inst. 59, 1231, 1977. 28. Fortner, G. W., and Kripke, M. L., J. fmmunol. 118, 1483, 1977. 29. Kripke, M. L., J. Reticuloendothel. Sot. 22, 217, 1977. 30. Fisher, M. S., and Kripke, M. L., Proc. Nat. Acad. ,‘j’ci. USA 74, 1688, 1977. 31. Howard, J. G., and Mitchison, N. A., Progr. Allergy 18, 43, 1975. 32. Gershon, R. K., and Kondo, K., Immunology 21, 903, 1971. 33. Fujimoto, S., Greene, M. I., and Sehon, A. H., J. Immunol. 116, 791, 1976. 34. Takei, F., Levy, J. G., and Kilburn, D. G., J. lmmunol. 118, 412, 1977.
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ULTRAVIOLET
82. 83. 84. 85. 86. 87. 88.
INFLUENCE
ON
THE
IMMUNE
SYSTEM
155
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