Proc. Nati. Acad. Sci. USAVol. 77, No. 7, pp. 4260-4264, July 1980Immunology

Serological survey of normal humans for natural antibody to cellsurface antigens of melanoma

(tumor immunology/melanocyte differentiation antigens/melanoma subsets)

ALAN N. HOUGHTON, MICHAEL C. TAORMINA, HISAMI IKEDA, TADASHI WATANABE,HERBERT F. OETTGEN, AND LLOYD J. OLDMemorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021

Contributed by Lloyd J. Old, April 22, 1980

ABSTRACT Sera of 106 normal adult men were tested forantibodies reacting with cell surface antigens of three estab-lished lines of cultured malignant melanoma. Positive reactionswith a protein A assay for IgG antibodies were extremely rare(1-2%). The frequency of positive reactions with assays for IgMantibodies was higher 5-15% in immune adherence assays and55-82% in anti-C3 mixed hemadsorption assays. After low-ti-tered sera and sera reacting with fetal calf serum components,conventional alloantigens, and widely distributed class 3 anti-gens were excluded, sera from seven individuals (one with IgGantibody and six with IgM antibodies) were selected for detailedanalysis. The serum containing the IgG antibody came from ahealthy 65-year-old Caucasian man; titers of antibody in hisserum ranged from <1/10 to 1/40,000 in tests with differentmelanoma cell lines. This IgG antibody identifies a differen-tiation antigen of melanocytes, provisionally designated Mel1, that distinguishes two classes of melanomas: 22 melanomacell lines typed Mel 1+ and 17 typed Mel 1-. Mel 1 is expressedby fetal fibroblasts but not adult fibroblasts and can be foundon a proportion of cultured epithelial cancer cell lines (5 out of23) but not on glioma or B-cell lines. The melanoma antigensdetected by the naturally occurring IgM antibodies are serolo-gically unrelated to Mel 1 but, like Mel 1, appear to be differ-entiation antigens that distinguish subsets of melanoma. TheseIgM antibodies detect antigens that are identical or closely re-lated to the AH antigen, a melanoma surface antigen that wasinitially defined by autologous antibody in a patient with mel-anoma. In view of the immunogenicity of both Mel 1 and theAH antigens in humans and their occurrence on more than 50%of melanomas, it remains to be seen whether antibody to theseantigens can be elicited by specific vaccination of seronegativemelanoma patients and whether this will have an influence onthe clinical course of the disease.

Despite considerable attention and effort, it remains unclearwhether immunological factors play any role in the patho-genesis of malignant melanoma. The major problem in re-solving this issue centers on the question of whether mela-noma-specific antigens exist and whether patients recognizethese antigens during the course of their disease. Although a vastliterature has accumulated on the subject of humoral and cel-lular immune reactions to melanoma, little in the way of criticalor convincing evidence for melanoma-specific antigens hasbeen forthcoming. What has been lacking in most studiesdealing with this question is proof that the reactions observedwere not due to immunity directed against HLA alloantigens,major or minor blood group antigens, or other classes of al-loantigenic differentiation antigens. To eliminate the need toconsider interference by alloantibodies, we turned several yearsago to an analysis of the immune reactivity of patients directedagainst cell surface antigens of their own tumors. The approachthat evolved in these studies, which we have termed autologous

typing, has now been applied to the study of several humantumor types-melanoma (1-3), astrocytoma (4), leukemia, (5),and renal cancer (6). Cultured lines of tumor cells (or cryo-preserved cells in the case of leukemia) serve as target cells thatcan be repeatedly tested for reactivity with autologous sera.Several serological techniques are used in parallel to detectdifferent classes of immunoglobulins, including complement-fixing antibodies. Positive reactions are then analyzed by ab-sorption tests with normal or malignant autologous, allogeneic,or xenogeneic cultured cells. In this way, three classes of cellsurface antigens detected by autologous typing have been de-fined. Class 1 antigens are restricted to autologous tumors andcan be found on no other cell type, normal or malignant. Class2 antigens are shared by some (but not invariably all) tumorsof similar derivation and may also be found on unrelated tumortypes, but have not been found on cultured normal cells. Incontrast to the restricted distribution of class 1 and class 2 an-tigens, class 3 antigens are widely distributed on cultured cells,both normal and malignant. The prototype class 1 antigen isthe AU melanoma antigen (1), a glycoprotein of approximately20,000-45,000 molecular weight that is unrelated to HLA an-tigen or 12 microglobulin (7). The prototype class 2 antigensare AH melanoma antigen (2), the serologically related AJ as-trocytoma antigen (4), and the AX and AG antigens of renalcancer (6). AH antigen is found on a large proportion of mela-nomas, all astrocytomas, and some sarcomas, but -not on anyepithelial cancers. Thus, autologous typing has permitted thedefinition of cell surface antigens that have a-high degree ofspecificity for cancer and that are immunogenic in their au-tologous host. Once class 1 and class 2 antigens are serologicallydefined, further analysis will provide insight into their structureand genetic origin.With the finding of serological reactivity to tumor cells in

autologous patients, the question that now needs to be answeredis whether the presence of antibody is a consequence of tumordevelopment or whether antibody and tumor are causally un-related. To investigate this, we have been analyzing the oc-currence and specificity of antibodies found in normal indi-viduals that react with surface antigens of tumor cells. Thepresent report deals with our study of natural antibodies tomelanoma cells.

MATERIALS AND METHODSTissue Culture. For the derivation of melanoma and other

cell lines, see refs. 1-6. Melanoma cell lines are designated SK(Sloan-Kettering), MEL (melanoma), and a series number, withthe coded initials of the melanoma patient given in parentheses.A culture of normal human epidermis (epidermis 390) waskindly supplied by Magdalena Eisinger. Two human fetal cell

Abbreviations: C3-MHA assay, anti-C3 mixed hemadsorption assay;IA assay, immune adherence assay; PA assay, protein A assay.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Table 1. Survey of sera from 106 normal adult men for artibody reactive with cell surface antigensof malignant melanoma

PA assay IA assay C3-MHA assayCell line Positive* Titer ranget Positive Titer range Positive Titer range

SK-MEL-28 (AU) 2/106 (1.9%) 256; 1280 6/106 (5.6%) 8-64 87/106 (82%) 8-128SK-MEL-13 (AH) 1/106 (0.9%) 256 10/106 (9.4%) 8-64 70/106 (66%) 8-128SK-MEL-29 (AV) 2/106 (1.90%) 256; 40,000 16/106 (15.1%) 8-64 58/106 (54.7%) 8-256

* >20% reactive target cells at serum dilutions 1/8 and 1/16.t 1/dilution of positive sera.

lines, Flow 4000 and Flow 5000, were purchased from FlowLaboratories (McLean, VA). B-cell lines from patients withmelanoma and renal cancer were derived from peripheralblood lymphocytes transformed by Epstein-Barr virus releasedfrom the B-95-8 marmoset lymphoid line (8). Melanocytes werecultured from postmortem specimens of adult human cho-roid.

Cultures were maintained in Eagle's minimal essential me-dium supplemented with 2 mM glutamine, 1% nonessentialamino acids, 100 Ag of penicillin per ml, 1 Ag of streptomycinper ml, and 10% (vol/vol) fetal bovine serum. In order to re-move heterologous fetal bovine serum components, melanomacells were cultured in screened 10% (vol/vol) human sera fromAB blood-type donors for at least 6 weeks (generally threesubcultures).

Cultures were regularly tested for mycoplasma, fungi, andbacteria, and contaminated cultures were discarded.

Sera. Sera were collected from healthy, nontransfused menand stored at -70'C. Age of the serum donors ranged from 19to 65 years.

Serological Procedures. The protein A hemadsorption (PA),immune adherence (IA) and anti-C3 mixed hemadsorption(C3-MHA) assays were performed as described (2-4, 6). Foranti-human IgG assays, indicator cells were prepared by con-jugating the immunoglobulin fraction of rabbit anti-human -Yheavy chain (DAKO, Copenhagen) to human O+ erythrocyteswith 0.005% chromium chloride. Three established melanomacell lines (SK-MEL-13, -28, and -29) were selected for initialserological screening. Assays were performed in Falcon 3040microtest II plates. Target cells (plated 2-4 days previously) andhuman sera (diluted 1/8 and 1/16) were incubated for 1 hr atroom temperature (PA and anti-human IgG assays) or at 40C(IA and C3-MHA assays). After the target cells were washed,indicator cells were added and incubated with target cells for1 hr (PA and anti-human IgG assays) or for 30 min (IA andC3-MHA assays). The plates were then washed gently, andreactions were evaluated under light microscopy. When cellsgrown in medium containing human serum were tested, 2%(wt/vol) gamma globulin-free human serum albumin (Sigma)in phosphate-buffered saline was substituted for the 5% gammaglobulin-free bovine serum regularly used in the serologicalassay. Qualitative absorption tests were performed by absorbing30 Al of serum (diluted according to the end point) with anequal volume of washed packed cells for 1 hr at room temper-ature (PA and anti-human IgG assays) or at 40C (IA and C3-MHA assays) and testing for residual antibody on target cellsgrown in Falcon 3034 plates (see ref. 6).

RESULTSReactions of Normal Human Sera with Cultured Mela-

noma Cells. Sera from 106 healthy, nontransfused male donorswere tested at a dilution of 1/8 and 1/16 against three mela-noma cell lines in PA, IA, and C3-MHA assays. All sera weretested on two separate occasions. The results of these tests aresummarized in Table 1. Reactivity was detected most fre-

quently in C3-MHA assays, considerably less frequently in IAassays, and only rarely in PA assays. Naturally occurring anti-bodies to A or B blood-group antigens did not contribute to theseresults; the target cells do not express A or B antigens and therewas no association between serum reactivity and ABO type ofthe serum donor.

Tests with Sera Reactive in PA Assays. Sera from 2 of the106 normal men (NS 74 and NS 124) had measurable antibodyto melanoma surface antigens detected by PA assays. NS 74reacted with the three melanoma target cell lines at titers of1/128-1/256. Antibody in NS 74 was directed against fetal calfcomponents adsorbed to the surface of the target cells; the re-activity of NS 74 could be absorbed by fetal calf serum, and NS74 had no reactivity with melanoma target cells grown inhuman serum rather than in fetal calf serum.NS 124 reacted with two of the three melanoma cell lines

with a titer of 1/40,000 on SK-MEL-29 cells, 1/640 on SK-MEL-28 cells, and <1/8 on SK-MEL-13 (Fig. 1). These titersremained unchanged in tests with target cells cultured inhuman serum. Parallel reactivity was demonstrable by usinganother assay for IgG antibody (anti-human IgG indicator cells),whereas NS 124 had no reactivity in the two IgM detectionsystems (IA or C3-MHA assays). Results with immunoglobulinfractions of NS 124 were consistent with the conclusion that themelanoma reactivity of NS 124 was due to antibody belongingto the IgG class.

Analysis of Melanoma Reactivity of NS 124. The donor ofNS 124 is a retired 65-year-old Caucasian man of Irish extrac-tion who has always enjoyed good health and has no allergies,recent immunizations, or family history of cancer. No suspiciousskin or soft tissue lesions were evident on physical examination.Titers of antibody reactive with SK-MEL-28 and SK-MEL-29remained constant in three serum specimens taken from thisindividual over an 18-month period. Immunoglobulin levelsin NS 124 were normal (IgG, 970 mg/100 ml; IgM, 200 mg/100ml; and IgA, 200 mg/100 ml) and serum immunoelectropho-resis revealed no abnormalities.

loorCA

._4:6aIG

0

50k

1/10 1/40 1/160 1/640 t 1/10,240 t1/2,560 1/40,960

Dilution of NS 124FIG. 1. Reactions of NS 124 with cell surface antigens of three

established melanoma cell lines in PA assays. 0, SK-MEL-29 (AV);a, SK-MEL-28 (AU); v, SK-MEL-13 (AH).

0- a W Ir 490 r--- v. -r - 7 L-

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4262 Immunology: Houghton et al.

Fig. 2 illustrates an individual absorption test with NS 124.Three melanoma cell lines absorbed reactivity for SK-MEL-29target cells (lower curves), whereas four other cell types (in-cluding another melanoma line, SK-MEL-37, and a Bcell linefrom the donor of SK-MEL-29) did not absorb reactivity (uppercurves). Table 2 summarizes the results of the absorption teststhat have been done. Twenty-two of 39 melanoma cell lines(56%) absorbed reactivity from NS 124, as did cultured fetalfibroblasts (derived from 16- to 20-week-old fetuses) and certainepithelial cancer lines. Adult fibroblasts, B-cell lines, a numberof epithelial cancer and glioma cell lines, and several types ofheterologous cells did not absorb NS 124 reactivity. The ca-pacity of different cell lines to absorb NS 124 reactivity forSK-MEL-29 target cells could be classified as strong, interme-diate, or weak, depending on whether absorption was completeor partial. Double absorptions with selected cell lines giving aninitial partial absorption resulted in complete absorption, in-dicating that quantitative (rather than qualitative) differencesin antigen expression accounted for differences in absorption

capacity. To examine this matter further, we determined thereactivity of NS 124 for different cell lines in direct PA tests(Table 3). Cell lines that absorbed NS 124 reactivity for SK-MEL-29 were also positive in direct PA tests. Conversely, celllines that had no absorbing capacity failed to react in directtests. Furthermore, antigen expression, as indicated by the titerof NS 124 in direct tests, correlated with the absorption capacityof cell lines. Cell lines with high antigen expression (1/640-1/40,000) showed complete absorption, whereas lines withlower antigen expression showed partial absorption.

Reactions of NS 124 with Cultured Melanocytes. Short-term cultures of melanocytes from adult choroid reacted withNS 124 in PA assays. (Tests with sera from 12 other normal in-dividuals were negative.) The titers of NS 124 in tests withmelanocytes from five individuals (6-10 days after in vitroculture) ranged from 1/100 to 1/200. Fibroblasts and epithelialcells, which could be distinguished in these cultures, were notreactive with NS 124. To determine the relationship betweenthe antigen detected on melanocytes and that on melanoma

Table 2. Analysis of PA reactivity of NS 124 for melanoma SK-MEL-29: Summary of absorption tests

Melanoma cellsSK-MEL-26 (AS)SK-MEL-28 (AU)SK-MEL-29 (AV)SK-MEL-41 (BH)MeWo (BI)SK-MEL-61 (CL)SK-MEL-63 (CN)SK-MEL-64 (CO)SK-MEL-78 (DC)SK-MEL-79 (DD)SK-MEL-86 (DK)SK-MEL-88 (DM)SK-MEL-89 (DN)SK-MEL-90 (DO)SK-MEL-91 (DP)SK-MEL-93 (DR)SK-MEL-95 (DT)SK-MEL-98 (DW)SK-MEL-101 (DZ)SK-MEL-44 (BT)SK-MEL-57 (CH)SK-MEL-100 (DY)

Positive absorption

Nonmelanoma cancer cellsME-180 (cervical)SK-RC-9 (renal)CaKi-1 (renal)BT-20 (breast)MCF-7 (breast)

Fetal cellsSkin fibroblasts F-1Skin fibroblasts F-3Brain fibroblasts F-2Lung fibroblasts F-2Choroid fibroblasts F-1Brain fibroblasts F-4Flow 4000 kidney fibroblastsFlow 5000 fibroblastsFetal brain F-20 (not

cultured)Fetal lung F-20 (not

cultured)

Negative absorption

Melanoma cellsSK-MEL-13 (AH)SK-MEL-19 (AL)SK-MEL-21 (AN)SK-MEL-23 (AP)SK-MEL-27 (AT)SK-MEL-30 (AW)SK-MEL-31 (AX)SK-MEL-33 (AZ)SK-MEL-36 (BC)SK-MEL-37 (BD)SK-MEL-40 (BG)SK-MEL-42 (BR)SK-MEL-52 (CC)SK-MEL-57 (CH)SK-MEL-69 (CS)SK-MEL-83 (DH)SK-MEL-87 (DL)

Nonmelanoma cancer cellsSK-OV-3 (ovarian)AJ (glioma)AS (glioma)BK (glioma)BL (glioma)MG 178 (glioma)U-138 (glioma)AA (retinoblastoma)Y-79 (retinoblastoma)Weli (retinoblastoma)Raji (Burkitt's)MOLT-4 (leukemia)J-82 (bladder)T-24 (bladder)SK-LC-LL (lung)SK-RC-1 (renal)SK-RC-2 (renal)SK-RC-6 (renal)SK-RC-7 (renal)SK-RC-10 (renal)SK-SC-1 (squamous)HT-29 (colon)

Nonmelanoma cancer cells(cont'd.)

SW-1222 (colon)SW-1116 (colon)SW-1006 (colon)CAMA (breast)AlAb (breast)ZR-75-1 (breast)MDA-MB-231 (breast)

EBV-transformedlymphocytes*

AH, AL, AV, AZ, CL,CO, CS, DE, DP

Fetal cellsFetal kidney epithelium

F-13Fetal liver F-20 (not

cultured)

Normal adult cellsAdult kidney epithelium 1Adult kidney epithelium 2Brain fibroblastsEpidermis 390

FibroblastsAD, AM, AQ, AR, AU,AV, BD, BF, CB, CS,DL, MeWo

Human erythrocytesA, B, AB, 0

Xenogeneic cellsVERO (monkey kidney)BHK (baby hamster kidney)B16 murine melanomaGuinea pig kidney, brain,

spleen, liver (not cultured)Sheep erythrocytes

* EBV, Epstein-Barr virus.

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Proc. Natl. Acad. Sci. USA 77 (1980) 4263

100r

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4.;

0404

._

bR

50 _

Z Z. 9 V

T r T1/2,000 t 1/8,000 t

1/4,000 1/16,000

Dilution of NS 124

FIG. 2. Absorption analysis of the PA reactivity of NS 124 formelanoma SK-MEL-29. Aliquots of NS 124 (diluted 1/200Q) were

absorbed with the indicated cell types. After removal of the absorbingcells, serum was tested for residual reactivity for SK-MEL-29 (AV)target cells by PA assays. (Upper curves) 0, Unabsorbed NS 124; &,AV Epstein-Barr virus-transformed B cells; A, sheep erythrocytes;*, SK-MEL-37 (BD); o, T24 bladder cancer. (Lower curves) v,SK-MEL-28 (AU); *, SK-MEL-86 (DK); 0, SK-MEL-29 (AV).

cells, we absorbed NS 124 with four melanoma cell lines ex-

pressing the NS 124 defined antigen and with four melanomacell lines not expressing the antigen. The results indicated thatthe antigen detected by NS 124 on normal melanocytes and

Table 3. Reactions of NS 124 with surface antigens of culturednormal and malignant human cells in PA assays

PositiveTiter

(recipro-Target cell cal)* Negativet

Melanoma cells Melanoma cellsSK-MEL-29 (AV) 40,000 SK-MEL-37 (BD)SK-MEL-26 (AS) 1,280 SK-MEL-13 (AH)SK-MEL-93 (DR) 1,280 SK-MEL-21 (AN)SK-MEL-28 (AU) 640 SK-MEL-19 (AL)SK-MEL-41 (BH) 640 SK-MEL-40 (BG)SK-MEL-61 (CL) 640 SK-MEL-31 (AX)SK-MEL-64 (CO) 640 SK-MEL-52 (CC)SK-MEL-79 (DD) 640 SK-MEL-30 (AW)SK-MEL-86 (DK) 640SK-MEL-98 (DW) 640 Nonmelanoma cancer cellsSK-MEL-57 (CH) 160 AlAb, AS glioma, T24,SK-MEL-100 (DY) 80 SK-RC-1, CAMA,

SW-1222, SW-1116,Nonmelanoma cancer cells HT-29SK-RC-9 320Me-180 160 Adult cellsBT-20 1,024 Epidermis 390, fetalMCF-7 640 kidney epithelium F-13

Fibroblasts: DL, AR, CX,Fetal cells CS, AU, BF, AD, AS

Choroid fibroblasts F-1 20Skin fibroblasts F-1 40Brain fibroblasts F-2 320Flow 5000 fibroblasts 40Brain fibroblasts F-20 320

* 1/dilution of NS 124 giving .20'% reactive target cells.t No reaction with NS 124 at a dilution of 1/10.

melanoma cells is either closely related or identical. In view ofthese findings, we have provisionally designated the antigenMel 1, to signify its characteristics as a melanocyte differen-tiation antigen and as an antigen defining a subset of mela-nomas.

Analysis of Sera Reactive in C3-MHA Assays. Sera from12 individuals with high-titered reactivity (1/64-1/128) againstSK-MEL-29 in C3-MHA assays were selected for furtheranalysis. (Because sera reactive by IA assays were invariablyreactive in C3-MHA assays, usually at higher titer, only C3-MHA reactions were studied.) Antibodies in six of the sera

identified fetal calf components (reactivity disappeared withmelanoma target cells grown in human serum) or widely dis-tributed class 3 antigens (reactivity disappeared after absorptionwith sheep erythrocytes or unrelated B-cell lines). The antigensdetected by antibodies in the remaining six sera were morerestricted, being expressed by certain melanomas and gliomasbut not by epithelial cancers. Absorption analysis of one of thesesera (NS 72) is shown in Table 4. The distribution of antigendetected by NS 72 was identical to the distribution of the AHmelanoma antigen (2) and the serologically related AJ astro-cytoma antigen (4) identified by autologous typing (Table 4).Absorption analysis of five other C3-MHA-reactive normal sera

has shown similar reactivity for the AH/AJ antigen system.

DISCUSSIONIn this initial survey of normal individuals for natural antibodyto cell surface antigens of melanoma, reactivity was most fre-quently demonstrable in C3-MHA tests, less frequently in IAtests, and only rarely in PA tests. Because our past experiencewith these tests indicates that the C3-MHA and IA assays detectIgM antibody primarily and the PA assay detects IgG antibody,it appears that IgM antibody is mainly responsible for the re-

Table 4. Absorption analysis of C3-MHA reactivity of NS 72 forSK-MEL-29 target cells: Comparison with the AH/AJ

phenotype of absorbing cellsAH/ AH/

Cell NS 72 AJ Cell NS 72 AJ

Melanomas B lymphocytes,SK-MEL-13 (AH) + + EBV-trans-SK-MEL-26 (AS) + + formed*SK-MEL-28 (AU) - - AH - -SK-MEL-29 (AV) + + AV - -SK-MEL-31 (AX) + + CO - -SK-MEL-61 (CL) - -SK-MEL-106 (EE) + + Fetal cellsSK-MEL-113 (EL) + + Flow 4000 - -

ChoroidalEpithelial cancers fibro-SK-RC-2 - - blastsSK-RC-9 - - F-1. - -SK-LC-LL - -ME-180 - - XenogeneicBT-20 - - cells

VERO - -GliomasAJ + + ErythrocytesAS + + HumanBE + + SheepU-373 MG + +

* EBV, Epstein-Barr virus.

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activity of normal human sera with melanoma surface antigens.To investigate this point further, sera from women, older in-dividuals, and individuals of different ethnic and geographicbackground must be tested with these assays and with otherserological tests to detect IgG antibody. Until such a detailedpicture of the reactivity of normal individuals to surface anti-gens of melanoma has been developed it will be difficult toplace in perspective the serological findings in patients withmelanoma, in individuals with other types of pigment disorders,or in members of families with a high melanoma incidence.The general method for determining the specificity of

melanoma antibodies found in normal individuals followsprocedures developed in our study of autologous reactivity tomelanoma cells in melanoma patients (autologous typing). Thefollowing comments summarize some of the salient points.

(i) Antibodies to components in fetal calf serum are notuncommon in human sera, and because these fetal calf com-ponents can adsorb to the surface of cells, sera containing suchantibodies will give positive reactions with cells grown in fetalcalf serum. As a rule, antibody to these antigens can be elimi-nated by absorption with fetal calf serum. However, a criticaltest to rule out reactivity due to fetal calf components is to growtarget cells in human sera for several passages and see whetherthe reactivity of positive sera persists.

(Ui) Alloantibodies to conventional HLA products can beexpected in pregnant or parous women and in transfused in-dividuals, and these can interfere with the detection of anti-bodies to other classes of surface antigens. For this reason, thesera in this initial survey came from normal men with no historyof blood transfusion. Methods are available, however, to ruleout reactions due to HLA antibodies, permitting the analysisof complex sera containing these antibodies. B-cell lineskransformed by Epstein-Barr virus) and lines of normal fi-*broblasts can be established from patients whose melanoma cellsare used as target cells in tests for natural antibody. If reactivityfor melanoma cells is not removed by autologous B cells or fi-broblasts, the presumption is that alloantibodies to HLAproducts are not involved. B cells have an advantage over fi-broblasts in this regard because B cells express HLA-D productsand these antigens are also expressed by melanoma cells (9, 10).Insofar as it has been tested, melanoma cells have not beenfound to express A or B blood-group antigens. With other typesof cancer, particularly epithelial cancers that do express theseantigens, absorptions with A and B erythrocytes are necessaryto evaluate the contributions of naturally occurring antibodiesto these antigens.

(iii) In addition to antibodies to fetal calf components, HLAproducts, and blood-group antigens, human sera contain anti-bodies that react with antigens that are widespread on culturedcells. These antigens, which we have referred to as class 3 an-tigens in autologous typing (3), are diverse and complex andhave not been studied in detail. Nonetheless, antibodies to mostclass 3 antigens can be easily removed by absorption with cul-tured cells, both normal and malignant, of autologous, allo-geneic, or heterologous origin.

Seven of the 14 sera that were analyzed in detail have reac-tivity that cannot be ascribed to antibodies against fetal calfcomponents, HLA antibodies, or widely represented class 3antigens. The antigen detected by IgG antibody in the sera ofone individual has the characteristics of a melanocyte differ-entiation antigen, and for this reason we have provisionallydesignated this antigen Mel 1. It is found on normal melanocytes

cultured from the choroid, but not on cultured adult fibroblasts,B cells, or T cells or on peripheral blood cells. Of the 39 lines ofmelanoma tested, 22 expressed Mel 1 and 17 did not, the latterpresumably being derived from progenitor cells in the mela-noblast - melanocyte lineage prior to the stage at which thisantigen is normally expressed. An alternative explanation forthis finding is that two separate pathways for pigment cell de-velopment are involved-e.g., melanocytic and nevocytic(11)-and that only those cells differentiating along one of thetwo pathways express Mel 1. Whatever the explanation, thedemonstration of two clearly defined melanoma subsets pro-vides the opportunity to see whether this difference correlateswith biological or clinical features of melanoma. Anothercharacteristic of this antigenic system is its presence on fibro-blasts of fetal origin but not on adult fibroblasts. Once again,this could indicate derivation from cells at different phases inthe same cellular lineage or derivation from two distinctlineages. Another possibility to account for the Mel 1- pheno-type of adult fibroblasts would be phenotypic repression of afetal trait in certain adult cells.

Analysis of the IgM antibodies detected by C3-MHA assaysindicates that the antigens identified by these antibodies areunrelated to Mel 1. Detailed study of one of these IgM anti-bodies shows that it detects an antigen with the same distribu-tion as the AH antigen of melanoma (2) and the serologicallyrelated AJ antigen of astrocytomas (4). A similar pattern ofreactivity has been found with five other naturally occurringIgM antibodies. Thus, the AH/AJ antigen represents a class ofdifferentiation antigens that has the potential for autoanti-genicity in normal individuals and in patients with cancer. (Thepossibility that natural antibody to AH/AJ antigen arises as aconsequence of infection with an oncogenic virus involved inthe etiology of melanoma and other AH/AJ+ tumors appearsunlikely, but cannot be excluded by present evidence.) Becausethe melanoma patient whose serum originally defined the AHsystem has had an unusually favorable clinical course, thepossibility exists that immunity to AH antigen is in some wayprotective. Attempts to raise AH antibody in melanoma patientsby deliberate immunization with AH+ cells are underway toevaluate this possibility.1. Carey, T. E., Takahashi, T., Resnick, L. A., Oettgen, H. F. & Old,

L. J. (1976) Proc. Natl. Acad. Sci. USA 73,3278-3282.2. Shiku, H., Takahashi, T., Oettgen, H. F. & Old, L. J. (1976) J. Exp.

Med. 144, 873-881.3. Shiku, H., Takahashi, T., Resnick, L. A., Oettgen, H. F. & Old,

L. J. (1977) J. Exp. Med. 145,784-789.4. Pfreundschuh, M., Shiku, H., Takahashi, T., Ueda, R., Ransohoff,

J., Oettgen, H. F. & Old, L. J. (1978) Proc. Natl. Acad. Sci. USA75,5122-5126.

5. Garrett, T. J., Takahashi, T., Clarkson, B. D. & Old, L. J. (1977)Proc. Natl. Acad. Sci. USA 74,4587-4590.

6. Ueda, R., Shiku, H., Pfreundschuh, M., Takahashi, T., Li, L. T.C., Whitmore, W. F., Oettgen, H. F. & Old, L. J. (1979) J. Exp.Med. 150,564-579.

7. Carey, T. E., Lloyd, K. O., Takahashi, T., Travassos, L. & Old,L. J. (1979) Proc. Nat!. Acad. Sci. USA 76,2898-2902.

8. Miller, G. & Lipman, M. (1973) Proc. Nat!. Acad. Sci. USA 70,190-194.

9. Winchester, R. J., Wang, C.-Y., Gibofsky, A., Kunkel, H. G.,Lloyd, K. 0. & Old, L. J. (1978) Proc. Nat!. Acad. Sci. USA 75,6235-6239.

10. Wilson, B. S., Indiveri, F., Pelligrino, M. A. & Ferrone, S. (1979)J. Exp. Med. 149,658-668.

11. Mishima, Y. (1967) Cancer (Philadelphia) 20,632-649.

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