Differentiation between B cells, T cells, and histiocytes in melanocytic lesions: Primary and metastatic melanoma and halo and giant pigmented nevi

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOCY

4,557-568

(I 975)

Differentiation Between B Cells, T Ceils, and Histiocytes in Melanocytic Lesions: Primary and Metastatic Melanoma and Halo and Giant Pigmented Nevi RICHARD A. LEE DELLON,’

L. EDELSON,’ VINCENT J. HEARING,’ MICHAEL FRANK,” EDMOND K. EDELSON.“.” AND IRA GREEN”

Dermatology’ and Surgery’ Branches, National Cancer Institute, and Laboratories of Clinical Investigation” and Immunology,’ National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland. and Dermatology Section, Clara Maass HospitaL4 Belleville, New Jersey Received

March

2X. 1975

Membrane properties of infiltrating mononuclear cells in melanocytic lesions were studied. In frozen sections of six primary melanomas and six halo nevi. the infiltrating cells lacked C3 receptors characteristic of B cells and IgG receptors characteristic of histiocytes. Viable infiltrating cells extracted from two primary melanomas formed rosettes with sheep erythrocytes. providing direct evidence of their T cell identity. In contrast, the majority of cells infiltrating melanoma metastases in four patients were identified as histiocytes by their IgG receptors and ultrastructural appearance. Focal aggregates of B cells and plasma cells were found in a giant pigmented nevus in areas of disrupted nevus cells. This suggests a B cell mediated response against nevus-associated antigens, possibly involving antibody dependent target cell lysis. It appears that a spectrum of cellular reactions to melanocytic lesions occurs in viva and that the type of infiltrating mononuclear cell is related to the type of lesion.

INTRODUCTION

Since the early observation by Handley (1) that areas of regression in malignant melanomas are associated with prominent lymphocytic infiltration, several histopathologic studies have further suggested a role for cell mediated immunity (CMI) in limiting the spread of melanoma. Nearly all primary cutaneous melanomas are infiltrated by mononuclear cells (2), an intense lymphocytic infiltration reportedly indicative of a good prognosis (3). Spontaneous regression of malignant melanoma has been well documented (4) and is associated with a particularly pronounced mononuclear infiltration of the lesions (5). In contrast, melanoma metastases are reportedly infrequently infiltrated by large numbers of mononuclear cells (6), suggesting a diminished host response. To date, efforts to identify the mechanism and effector cells of CM1 in melanoma patients have involved study of in rifro parameters. Lysis of melIi Reprint requests Columbia University

should School

be sent to Dr. Richard L. Edelson, Department of Dermatology, of Medicine, 630 W. 168th Street. N.Y.. N.Y. 10031 557

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

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anoma target cells by sensitized lymphocytes and activation of immune lymphocytes following incubation with melanoma cells or melanoma cell extracts have been reported (reviewed in reference 7). Each of these in \‘ifro reactions can be affected by blocking and unblocking serum factors. Thymus-derived (T) cells t 8) and macrophages (9) have been reported to serve as anti-melanoma effector cell\ in these in vitro systems. Since it is now clear that each of the three major groups of mononuclear cells (T cells, bone marrow-derived or B cell5 and macrophages) are capable of lysing target cells in vitro (and presumably ir! rir’ol by mechanisms distinctive to their cell type and requiring effector cell-target cell contact (1 O), direct identification of specific infiltrating cell populations in lesion\ is a prerequisite to the establishing of the clinical relevance of irl t,itr-o findings. Recently, we have demonstrated that it is possible to distinguish between B cells, T cells. and cells of the macrophage series (monocytes, histiocytes! in human cutaneous infiltrates ( I 1). We report here our findings from the appIica-tion of that method to the study of infiltrates in primary and metastatic meianoma lesions. in halo nevi, and in a giant pigmented nevus. These observations may allow a correlation between histologic appearance of melanocytic lesion\ and the several proposed mechanisms for in \,itro target cell destruction. MATERIALS

AND METHODS

Patients Melanocytic lesions from 14 patients were studied. Six of these were primary melanomas: of these, four were classified as superficial spreading, and two were classified as lentigo maligna melanoma (Hutchinson’s melanotic freckle) by standard histopathologic criteria ( 13,). Metastatic melanoma lesions from four other patients, halo nevi from three patients, and one giant pigmented nevus also were investigated. None of these patients received immonosuppressive therapy prior to or during this study. Identijication

of’ Subpopulations

oj’ Mononuclrrv

C’elis in Lrsiom

Excised tissue was treated as previously described ( 11). Briefly. 6 pm unfixed frozen sections were layered with sheep erythrocytes containing on their surfaces moieties which would specifically interact with receptors on the surfaces of mononuclear cells. Sheep erythrocytes (E) bearing a surface coat of IgM antiForssman antibody (A) and complement (C) to form IgM EAC complexes adhere to B cells and some histiocytes through receptors those cells have for the cell bound fragment of the third component of complement (C3). IgG EA adheres to histiocytes in frozen section via receptors those cells have for IgG antibody. Although both B cells and some histiocytes bind IgM EAC. only histiocytes bind IgG EA in our system, permitting a distinction between the two types of cells. Neither IgM EAC or IgG EA adhere to T cells since these cells lack both the C3 and IgG receptors. In our laboratory, the T cell receptor for uncoated E has to date not been identifiable in frozen sections. Two sets of controls were used. First, IgM EA will not specifically adhere to any mononuclear cells in section because of the absence on this red cell reagent of both comple-

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ment and IgG; this reagent was therefore routinely used to rule out nonspecific adherence of the marker erythrocytes. Second, normal human lymph nodes or either normal human or mouse spleen were run in parallel with test tissue sections as a positive control. IgM EAC will adhere to the B cells which largely constitute the lymphoid follicles but not to the T cell interfollicular zones in lymph nodes or to the T cell periateriolar region in the splenic white pulp. IgG EA will adhere to the histiocytes of both the splenic white pulp and the medullary sinuses in lymph nodes. In addition, sufficient tissue (at least 2 cm{) was available from two patients with primary melanoma to process as follows: unfixed excised tissue was finely minced, filtered on a Ficoll-Hypaque gradient to obtain a suspension of viable infiltrating cells and studied as previously described in detail elsewhere (11, 13). Briefly, the percentage of T cells in the resulting suspension was determined by counting the numbers of rosetted and unrosetted mononuclear cells following incubation with neuraminidase pretreated sheep erythrocytes. Morphologic Studies Surgically obtained material was prepared for study as follows. For routine histopathologic study by light microscopy, specimens were fixed in formalin. paraffin embedded, and stained with hematoxylin and eosin. For electron microscopy, the specimens were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 hr at 4°C. After washing overnight in 0.1 M phosphate buffer with 0.1 M sucrose at 4”C, the specimens were postfixed in 1% osmium tetroxide for 2 hr at 4°C. The tissues were then dehydrated in a graded series of ethanol, with propylene followed by propylene oxide, and were then infiltrated oxide: epon (1 : 1 and 1 : 3, v : v). Following epon embedding, sections were cut on a Porter Blum MT2 ultramicrotome, strained with uranyl acetate and lead citrate, and viewed in a Siemens IIA electron microscope. Histochemicai

Localization

of Tyrosinase

After the initial fixation in glutaraldehyde, the specimens were washed thoroughly in buffer and then were incubated in 0.1% dihydroxyphenylalanine (DOPA) in buffer for 1 hr at 37°C. They were then postfixed in osmium tetroxide and processed as detailed above. Controls for this reaction included substrate (DOPA) deficient in incubation medium and preincubation with the inhibitor diethyldithiocarbamate (10 n-N) by the method of Okun et al. ( 14). RESULTS

Localization

of B Cells, T Cells, and Histiocytes

in Lesions

Frozen sections of normal spleens or lymph nodes were always run as controls because of the known compartmentalization of these tissues into B cell, T cell, and histiocyte predominant regions (15). In these control tissues, as reported previously (1 I), IgM EAC adhered strongly to the B cells in the lymphoid follicles while IgG EA adhered to the histiocytes in splenic red pulp. IgM

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FIG. I. Section of primal-y melanoma. IgM EAC or IgG EA. T cell identity rosettes with sheep erthrocyteh (insert)

Infiltrating small lymphocytes of the infiltrating cells wa& after removal from unfixed

the

rosette

i\ identified

as a lymphocyte.

Hematoxylin

ET

AL..

and

ewin

~art-ows~ I’~~ilcd tu hind CI~IICI confirmed by their fwmation 01 tissue. The ccl1 in the ccntri. 01 stain.

(Main

figure

\ 2.50.

lnsclt

’ I 000.)

EA. used as an additional control, consistently failed to adhere to mononuclear~ cells in frozen sections. further demonstrating the specificity of the reagents. Routine histopathologic preparations from all the primary melanomas demonstrated significant mononuclear cell infiltration comprised largely of small lymphocytes (Fig. I). In frozen sections from lesions from each of these patients. the large majority of the infiltrating cells failed to bind either IgM EAC (B cell and histiocyte reagent) or IgG EA (histiocyte reagent), suggesting that these cells were neither B cells or histiocytes. Since on repeated attempts we have been unable to demonstrate specific binding of sheep erythrocytes to the nonviable T cells in frozen sections but can readily identify viable T cells by their formation of rosettes with the same reagent ( 1 I ), the two largest primary lesions (from patients 2 and 3) were finely minced and filtered to obtain suspensions ot viable infiltrating cells. These cells were then incubated with sheep erythrocytcx. the percentage of rosette forming cells were determined: and rosettes wet-e then settled on glass slides and stained with giemsa to identify the cells in their centers (insert Fig. 1). The mononuclear cells could be readily distinguished from the neoplastic cells and normal epithelioid elements. Eighty-five percent of the mononuclear cells extracted from the lesion from patient 2 and 9 1% of those from the lesion of patient 3 formed rosettes with sheep erythrocytes. confirming the impression from frozen sections that the large majority of infiltrating cells

INFILTRATING

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TABLE

SURFACE

RECEPTORS

OF INFILTRATING

MELANOMAS

AND

1 MONONUCLEAR Receptors

Tissue

source

561

NEW

Diagnosis

IgM

CELLS” on cells in section”

EAC

IgG

EA

Control normal lymph nodes Control normal spleens Control lymph node from patient with chronic B cell leukemis Patient 1 Patient

2’

Patient

3”

Patient

4

Patient

5

Patient

6

Patient

7

Patient

8

Patient

9

Patient

10

Patient Patient Patient Patient

11 12 13 14

Primary melanoma (lentigo meligna melanoma) Primary melanoma (lentigo meligna melanoma) Primary melanoma (superficial spreading melanoma) Primary melanoma (superficial spreading melanoma) Primary melanoma (superficial spreading melanoma) Primary melanoma (superficial spreading melanoma) Metastatic melanoma (melanotic and amelanotic) Metastatic melanoma (melanotic) Metastatic melanoma (melanotic) Metastatic melanoma (melanoma) Halo nevi (3) Halo nevi (2) Halo nevus Giant pigmented nevus

Follicles reactive Follicles reactive Entire section reactive

Medullary sinuses reactive Red pulp reactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Nonreactive

Both types nonreactive Nonreactive

Melanotic reactive, amelanotic nonreactive Reactive

Nonreactive

Reactive

Nonreactive

Reactive

Nonreactive Nonreactive Nonreactive Reactive

Nonreactive Nonreactive Nonreactive Nonreactive

I’ E = Sheep erythrocytes. A = Anti-Forssman antibody. h Reactive = Infiltrates in which 10% infiltrating cells bind r 85% of mononuclear cells extracted from lesion formed d 91% of mononuclear cells extracted from lesion formed

Nonreactive

C = Complement. reagent. rosettes with E. rosettes with E.

were T cells. The tissue obtained from each of the other primary melanoma lesions was too small for similar testing. These results are summarized in Table 1. In contrast to what was observed in the primary melanomas, only sparse lymphocytic infiltration was evident in metastatic melanoma lesions from four other patients. Patient 7 was of particular interest because of the presence of both melanotic and amelanotic metastases. Although the amelanotic metastases in that patient were grossly and by light microscopy unpigmented, we demonstrated that they contained tyrosinase activity and were therefore identifiable as

569

FIG. DOPA. tyrosinase.

this

EDELSON

2. Electron Previously Lead

micl-ogl-aph unmelanized citrate

FIG. 3. Section from reagent. indicating

and

edge that

of melanoma melanosomes uranyl

acetate

cell

E’I-

AL.

in amelanotic become evident

mctartasli (arrow\)

I’<>llowing inLuhatio!l hecauzr of the prewncc

\I 1111 <)I

TV 3 1,000).

of melanotic metastabih treated they are histiocytes. Hematoxylin

with and

IgG EA. Most infiltrating eobin stain (X 250).

cells

hind

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NEVI

melanoma metastases (Fig. 2). As indicated in the table and shown in Fig. 3, each of three pigmented cutaneous metastases in patient 7 contained foci of marked histiocytic infiltration (IgG EA positivity), a feature also noted in the study of the pigmented metastases from the other three patients. That these particular lesions did in fact contain large numbers of phagocytic cells was confirmed by electron microscopic examination: melanophages and malignant melanocytes could readily be distinguished from one another since the former contained large packets of membrane bound melanosomes while the latter contained individually dispersed melanosomes. However, neither of two amelanotic metastases in patient 7 contained significant numbers of IgG EA positive cells. suggesting a relationship between melanogenesis by the tumor cells and the induction of histiocytic infiltration. Halo nevus is a descriptive term applied to spontaneously regressing pigmented moles which characteristically have depigmented halos around partially resolved nevi. These lesions are frequently accompanied by a dense inflammatory infiltrate (16-18). Inclusion of halo nevi in this study was motivated by the suggestion that in at least certain instances they may result from an immunologic reaction against melanoma or related antigens (19, 20). The halo nevi in one of the three patients occurred after removal of a primary melanoma while the lesions of the other two individuals occurred in the absence of any clear histopathologic evidence of malignant change. Two halo nevi and one clinically

FIG. straight same (thick

4. Section arrows)

of giant bind this

lesion (insert) arrows). Both

reveals figures

pigmented nevus reagent indicating presence hematoxylin

treated with IgM EAC. Infiltrating that they are B cells. Paraffin-embedded

of numerous and eosin

plasma stained

cells (curved (X 250).

arrows)

lymphocytes section and

melanophages

(thin from

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normal compound nevus were studied from each of these patients. All of the halo nevi contained a mixed inflammatory cell infiltrate consisting primarily at small lymphocytes with scattered melanophages. The lymphocytic cells in sections of these halo nevi had the same binding properties as those lymphocytes found in the primary melanomas from patients l-6 as judged by their failure to bind either IgM EAC and IgG EA. Melanophages in those lesions could be identified both by the presence of large melanin packets and their binding of Ig(; EA in frozen sections. The clinically normal compound nevi from each of the three patients with halo nevi did not contain infiltrating cells in significant number. A significant percentage of giant pigmented nevi are reported to under-g<> malignant degeneration (3 l-14). One such nevus. covering the entire back of an adult individual (patient no. 14), was therefore excised in three stages as prophylaxis against melanoma. Deep in the dermal part of the nevus and in pigmented projections extending into back musculature, numerous focal accumulations of mononuclear cells were seen abutting on nevus cells which appeared benign by, both light and electron microscopy (Fig. 4). These infiltrates were comprised mainly of B cells, as judged by the fact that they bound IgM EAC and also contained some plasma cells (Fig. 5).

5. Electron

micrograph

of

pigmented

nevus. Portions of three melanocytic cells are somes. An adjacent melanophage is identified melanosome\ surrounded by membranes, Lead

cell\

from

area

of

I3 cell

infiltrate

of giant

seen (straight arrows) with singly disperxd by the presence of large aggregate5 citrate

and

uranyl

acetate

(k 3500)

pigmented melanuof ingested

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DISCUSSION A wide diversity of host anti-melanoma cell mediated effector mechanisms have been detected in vitro by testing blood lymphocytes (in the presence or absence of serum factors) against cultured autologous or allogeneic melanoma cells. Lymphocyte mediated destruction of these target cells has been identified in a number of independent laboratories (Z-33). Many patients with melanoma have serum factors which abrogate this in r*itro reactivity, and the presence of these factors appears to be related to the extent of disease (34, 35). Earlier discrepancies in results from different laboratories now seem to have been related to variation in experimental design: preincubation of blocking serums with target tumor cells interfered with lymphocyte mediated cytolysis apparently only when antibody was complexed with tumor antigen (36, 37), while serum containing either free tumor antigen or antigen complexed with antibody (presumably in antigen excess) was capable of inhibiting the reaction if first incubated with the effector cells (38). Serum factors, possibly additional anti-tumor antibody, present in the serums of melanoma-free patients canceled the blocking effects of other serums (39). Efforts to establish the identity of the effector cells in these in vitro systems in which melanoma cells are used as targets have implicated both T cells and cells of the phagocyte series. Wybran et al. (8) recently demonstrated that a T cellenriched population of mononuclear cells had increased cytotoxic activity against cultured allogeneic melanoma cells, while Mitchell et al. (9) found that several melanoma patients had serum factors which could “arm” heterologous macrophages to bind to allogeneic melanoma cells. Furthermore, the frequency with which melanoma patients have serum antibodies against tumor antigens (26, 40-46) indicates that precursor B cells with anti-melanoma specificity are also present in these patients. The relevance of these in vitro observations to possible in t,itfo antitumor cell mediated immunity can be determined through identification of the types of mononuclear cells actually infiltrating lesions, and this was the purpose of the present study. Our results indicate that the identity of the majority of infiltrating cells de-. pends. at least in part, on the type of melanocytic lesion. In each of six primary melanomas and six halo nevi, the infiltrating cells lacked receptors characteristic of B cells and histiocytes. In two of the primary melanomas, it was possible to bring many of the infiltrating cells into suspension and more definitely determine that they were T cells, as judged by their ability to bind sheep erythrocytes. In contrast, histiocytes were the predominant infiltrating cells in the pigmented melanoma metastases, while B cells and plasma cells were seen infiltrating a giant pigmented nevus. The findings that T cells were infiltrating the primary melanomas support an important defensive role for T cells. Lesions at any one of several levels of cell function could explain the absence of significant numbers of T cells from metastatic lesions. In addition to an inhibition by serum blocking factors (34-38) and a generalized decrease in the size of the T cell pool (37) in such individuals, more central defects could exist. Substantial evidence from experimental systems indicates that at least two populations of T cells are involved in the generation of

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cytotoxic effector cells (48. 49): abnormalities localized to one of these ~1. ceil subpopulations could result in a failure to mobilize lymphocytes. Conversely. metastatic melanoma cells may not express certain tumor antigens present in primary lesions. Such an occurrence was noted in a murine sarcoma system by Faraci rf al. (50). It is not clear whether the histiocytic infiltrates in the metastatic melanomas 111 this series represent specific or nonspecific host response. Evans et LI/. (5 I-53 ) have shown in a murine tumor system that macrophages can be “armed” by soluble factors and thereby gain specificity as effector cells. and Mitchell et 01. (9) identified serum factors in melanoma patients which “armed” heterologouk macrophages so that they could specifically bind to cultured allogeneic melanoma cells. However, one of our patients had both melanotic and amelanotic metastases, but only the melanotic metastases were infiltrated by histiocytc\ Although this might reflect diminished antigenicity of the amelanotic metastase\. it could also result from a phagocyte chemotactic property of melanin granule\. as suggested by the findings of Lejeune (54). The observation that in one patient a giant pigmented nevus was infiltrated by B cells (lymphocytes bearing C3 receptors) and by plasma cells is particularly intriguing. Although such lesions have a tendency to undergo malignant change. spontaneous infiltration by mononuclear cells has not been previously reported (21-24, 55). If the focal nature of the infiltrates in our patient represents ;: cellular response to newly expressed antigens on nevus cells undergoing malignant change. the neoplastic alteration is quite subtle since the cells even at the infiltrated sites are morphologically normal. However. the destruction of melanocytic cells at those sites (as determined by the presence of phagocytic cells containing many melanosomes) suggests that B cells and/or plasma cells were involved in immunologic attack against the nevus cells locally. Our observation that B lymphocytes from this same patient are specifically stimulated by autologous nevus extract7 is added evidence that some of his B cells have been specifically sensitized to nevus associated antigens. It is tempting to speculate that the mechanism involved is the antibody-dependent (AD) lymphocyte mcdiated cytotoxicity first described by Moller (56) whereby target cells coated bk specific antibody are lysed even by uncommitted lymphocytes. Direct contact I\ required between effector and target cells (57): the effector cells are not T cell\ (58) and have receptors for the Fc fragment of IgG through which binding occurs to target cells (59. 60): and cells carrying (‘3 receptors are known to participate in AD lysis (61). The only previously described human tumor system in which AD Iysis has been suggested by in 19if~ofindings has been the transitional cell carcinoma of the bladder (62-64). Our observations suggest that such an ef. fector mechanism may be operative in the giant pigmented nevus studied. Plasma cells in the infiltrates might be producing IgCi antibody against newly expressed membrane antigens on certain altered nevus cells. and this locally produced antibody could facilitate AD lysis by the adjacent B cells. .A particularly efficient system would exist if the plasma cells were differentiating directly i Edelson.

K. and

Dean.

J.. manuscript

in prrparation.

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from the infiltrating B cells. It will be important to determine whether giant nevi from other patients also contain abundant B cells and plasma cells. It appears from these studies that in different clinical settings B cells, T cells, and histiocytes may each play important rules in the host response to melanocytic lesions. Distinction between the different types of mononuclear cells in such lesions not only provides important clues as to what effector mechanisms are operating in viva but may be valuable in correlating in vitro assays with in vivo events in the evaluation of the efficacy of various forms of therapy. ACKNOWLEDGMENTS The authors are grateful to Dr. Marvin A. Lutzner, Chief of the Dermatofogy Branch of the National Cancer Institute. for his support and helpful suggestions during the course of these studies, to Mrs. Virginia Farkas for help in the preparation of the manuscript, and to Mr. Harold Schaefer for photographic assistance.

REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Handley, W. S., Lancet 1, 927, 1907. Couperus, M., and Rucker, R. C., Arch. Dermatol. Syph. 70. 199, 1954. Thompson, P. G., Pigment Cell 1, 285, 1973. Bodenham, D. C., Ann. Roy. Coil. Sug. Eng. 43, 2 18. 1968. Lloyd, 0. C.. Proc. Roy. Sot. Med. 62, 543, 1969. Payan, H. M., Gilbert, E. F., and Jacobs. W. H., South Med. J. 63, 1350, 1970. Hellstrom. K. E., and Hellstrom, I., Advan. Immunol. 18, 209, 1974. Wybran, J.. HeIlstrom, I., Hellstrom, K. E., and Fudenberg, H. H., fnt. J. Cancer 13, 515, 1974. Mitchell, M. S., Mokyr, M. B., Aspnes, G. T., and McIntosh, S.. Ann. fnt. Med. 79, 333, 1973. Cerottini. J. C., and Brunner, K. T., Advan. Immunol. 18, 67, 1974. Edelson, R. L., Smith, R. W., Frank, M. M., and Green, I.. J. Invest. Dermatol. 61, 82, 1973. McGovern, V. J.. Mihm. M. C.. Bailly, C., Booth, J. C., Clark, W. H., Cochran, A. J., Hardy, E. G., Hicks, J. D., Levene, A., Lewis, M. G., Little, J. H.. and Milton, G. W., Cancer 32, 1446, 1973. Edelson. R. L., Kirkpatrick, C. H., Shevach, E. M., Schein, P. S.. Smith, R. W., Green, I., and Lutzner, M., Ann. Int. Med. 80, 685, 1974. Okun, M. R.. Donnellan, B., and Pate], R., Lab. Invest. 27, 15 1. 1972. Craddock. C. G., Longmire, R., and McMillan, R., N. Engl. J. Med. 285, 324. 1971. Sutton. R. L., J. Cutan. Dis. 34, 797, 1916. Kopf, A. W., Merrill, S. D., and Silberberg, I., Arch. Dermatol. 92, 14, 1965. Wayte, D. M., and Helwig, E. B., Cancer 22, 69, 1968. Copeman, P. W. M.. Lewis, M. G., Phillips, T. M., and Elliott, P. G., &it. J. Dermarol. 88, 127, 1973. Epstein. W. L., Sagebeil, R., Spitler, L., Wybran, J., Reed, W. B., and Blois, M. S.. J. Amer. Med.

2 I. 22. 23. 24. 25. 26.

Ass. 225, 373,

1973.

Pack, G. T., and Davis, J., Surgery 49, 347. I96 I. Reed, W. B., Becker, S. W., Becker. S. W., and Nickel. W. R., Arch. Dermatol. 91, 100. 1965. Penman, H. G., and Stringer, H. C. W., Arch. Dermatol. 103, 428. 1971. Kaplan, E. N., Plast. Reconstr. Surg. 53, 421, 1974. Hellstrom, I., Hellstrom, K. E., Sjogren, H. O., and Warner, G. A., Int. J. Cancer ‘7, 1, 1974. Fossati, G., Calnaghi, M. I., Della Porta, G., Cascinelli, N., and Veronesi, U., Int. J. Cancer 8, 344, 1971. 27. Jagarlamoody, S. M., Aust, J. C., Tew, R. H., and McKhann. C. F.. Proc. Nat. Acad. Scj. 68, 1346, 1971. 28. Nagel, G. A., Piessens, W. F.. Stilmant, M. M., and Lejeune, F., Eur. f. Cancer 7, 41, 197 I. 29. De Vries. J. E.. Rumke, P.. and Bernheim, J. L., Int. J. Cancer 9, 567, 1972.

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30. Mackie. R. M., Spilg, W. G. S.. Thomas. c‘. E.. and Cochran. A. J., Brit. J. Drrmur~(. 87, i”_i. 1972. 31. Clark. D. A.. and Nathanson, L.. Pign~ent Cell 1, 350. 1973. 32. Byrne. M.. Heppner, G., Stolbach. L.. Cummings, F.. McConough. E., and Calabresi. P.. ,Yur. Cancer Inst. Monogr. 37, 3, 1973. 33. Levy, N. L.. Nat. Cancer. Inst. Monogr. 37, 85. 1973. 34. Hellstrom. 1.. Sjogren. H.. Warner, G.. and Hellstrom, K.. fnt. .I. Cancer 7, 226. 197 I, 35. Currie. C., Brit. J. Cancer 28, 153 (Suppl. 11, 1973. 36. Sjogren, H. 0.. Hellstrom. I. Bansal. S. C.. and Hellstrom. K. E.. Proc. Nut. A~trd. S(i 6X. 1372. 1971. 37. Sjogren, H. 0.. Hellstrom. I.. Bansal. S. C.. Warner. G. A., and Hellstrom, K. E.. Int. ./. (‘trncer 9, 274. 1973. 38. Currie, G. A.. and Basham, C.. Brit. J. Cancer 26. 427, 1971. 39. Hellstrom. I., Hellstrom. K., Sjogren, H.. and Warner. G., Int. J. Cancer 8, I85? 1~71. 40. Morton. D. L., Malmgren. R. A., Holmes. E. C., and Ketcham, A. S.. Surgery 64, 233, 106X. 41. Lewis. M. G.. Ikonopisov, R. L., Nairn. R. C.. Phillips. T. M., Fairley. G. H., Bodenham. D. C.. and Alexander, P., Brit. Med. J. il. 547, 1969. 41. Lewis, M. G., and Phillips, T. M., J. Nat. Cancer Inst. 49, 915, 1972. 43. Bourgoin. J. J.. and Bourgoin. A.. Pigment Crll 1, 366. 1973. 44. Cox, I. S.. and Ronisdahl, M. M.. Pigment Cell 1. 372. 1973. 45. Mukherji, B.. Nathanson, L.. and Clark, D. A.. Yale ./. Bit>/. Med. 46, 681, iY73. 46. Lewis. M. G.. Avis. P. J. G.. Phillips. T. M.. and Sheikha. K. M. A., Yale J. Bio/. Med. 46, 661. 1973. 47. Wybran. J.. and Fudenberg, H. H.. J. C/in. Invest. 52, 1026, I Y73. 48. Hayry, P.. Anderson. L. C., Nordlung. S., and Virolainen. M., Transplant. Rev. 12, Y I, 1~72. 49. Tigerlaar, R. E.. and Asofsky, R.. J. Exp. Med. 135, 1059, 1972. 50. Faraci, R. P.. Schour. L.. and Lesser, C. R.. Cancer 9, 1974. 51. Evans. R., and Alexander, P.. Nature (London) 236, 168. 1972, 52. Evans, R.. and Alexander. P., Immunology 23, 6 IS. 1972. 53. Evans, R., and Alexander, P., immunology 23, 627, 1972. 54. Lejeune. F. J., Yale .I. Biol. Med. 46, 368. 1973. 55. Mark, G. J., Mihm. M. C., Litoplo, M. G.. Reed, R. J.. and Clerk. W. H.. Human Puthol. 4, 395. 1973. 56. Moller. E., Science 147, 873. 1965. 57. Perlmann, P., and Holm, G.. Advun. Immunol. 11, 1 17. 1969. 58. Wigzell. I-1.. Goldstein. P.. Svedmyr, E. A. J.. and Jondahl. M.. Transplant. Proc~. 4, 3 I I. 1972. 59. MacLennan. 1. C. M.. Loewi, G., and Harding, B., lmmuno[ogy 18, 397. 1970. 60. MacLennan, 1. C. M., Clin. Exp. lmmuno/. 10. 175, 1972. 61. Van Boxel. J. A.. Paul, W. E., Frank, M. M., and Green, I.. J. lmmuno/. 110, 1077. 1973. 62. O’Toole. C., Stejskal. V., Perlmann, P.. and Karlsson. M., J. Exp. Med. 139, 457. 1974. 63. O’Toole, C.. Perlmann. P.. Wigzel, H.. Unsagaard, B., and Zetterbind, C. G.. Lancer 1, 10x5. 1973. 64. Hakala. T. R.. and Lange. P. H., Science 184. 79s. 1974.