antigens in gynaecological neoplasms: An immunohistological

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1 Br. J. Cncer (1985), 52, Expression of MHC products nd leucocyte differentition ntigens in gynecologicl neoplsms: An immunohistologicl nlysis of the tumour cells nd infiltrting leucocytes A. Ferguson', M. Moore2 & H. Fox3 Deprtments of 'Obstetrics nd Gynecology; 3Reproductive Pthology, St. Mry's Hospitl, Mnchester M13 OJH; 2Deprtment of Immunology, Pterson Lbortories, Christie Hospitl nd Holt Rdium Institute, Mnchester M2 9BX, UK. Summry Monoclonl ntibodies directed ginst monomorphic determinnts of Clss I nd Clss II products of the mjor histocomptibility complex (MHC) nd ginst leucocyte differentition ntigens were used in n indirect immunoperoxidse technique to compre their expression in norml nd mlignnt disese of the ovry, cervix nd endometrium. MHC Clss I products, strongly expressed on norml ovrin epithelium, were uniformly bsent from 7/8 ovrin crcinoms of vrying histology. Lck of Clss I expression ws lso feture of 6/1 cervicl crcinoms nd of 4/8 endometril crcinoms, in comprison with their repsective norml tissues. Reltive to norml tissue epithelium MHC Clss II products, could be either lost or gined, the pttern of expression being either uniform or heterogeneous. Leucocytes were sprse in norml ovry but more numerous in cervix nd endometrium. In tumours, with few exceptions, they were bundnt, though usully confined to the strom. T cells, lrgely of cytotoxic/suppressor (OKT8) phenotype, tended to predominte though in some tumours, prticulrly cervicl crcinom, lrge numbers of mcrophges nd to lesser extent, B cells, were sometimes detected. By contrst, leucocytes of nturl killer (NK) phenotype were virtully non-existent in ny tumour or norml tissue. The ingress of leucocytes into gynecologicl neoplsms does not pper to be rndom event nd my be evoked by n immune response ginst tumour-ssocited ntigens. However, the reltionship between in situ mononucler cell infiltrtion nd MHC expression on epithelil tumour cells is complex nd remins to be elucidted. It is now widely recognised tht the biologicl behviour of tumours, which my differ mrkedly for neoplsms of similr histopthologicl ctegory nd grde, is determined to significnt extent by interctions with neighbouring cells nd by their environment in generl. Attempts to unrvel the criticl fctors in these complex in vivo interctions hve, in prt, focussed on the reltionship between leucocyte infiltrtion nd prognosis, for which, there exists, t lest for some neoplsms, positive correltion (Underwood, 1974; lochim, 1976) s well s upon the nti-tumour properties in vitro of inflmmtory cells recovered from disggregted neoplsms (see Hskill, 1982; Moore, 1984; Vose & Moore, 1985). In the study of humn tumours, only limited exmintion of the mny prmeters involved in leucocyte ingress is presently fesible minly becuse the ntigens expressed by these neoplsms hve only been prtilly defined. Of theoreticl relevnce to host recognition of tumour cells in this context, is the expression of products of the mjor Correspondence: M. Moore. Received 15 April 1985; nd in revised form, 9 July histocomptibility complex (MHC). Clss I (HLA- A, B, C) ntigens re found on virtully ll norml epithelil cells, nd their recognition is essentil for the killing of virus-infected trget cells (McMichel, 1978) by cytotoxic T lymphocytes nd possibly of tumour cells lso. MHC Clss II products were until recently, considered to be restricted to cells of the immune system, including B lymphocytes, mcrophges, vsculr endothelil, dendritic nd other ntigen presenting cells, s well s ctivted T lymphocytes (Ko et l., 1979). Now in ddition, certin epitheli nd significnt number of nonlymphoid neoplsms including those of brest, colorectl crcinom nd mlignnt melnom re known to disply Clss II molecules (Ntli et l., 1981; Thompson et l., 1982; Whitwell et l., 1984; Dr & Fbre, 1983; Rognum et l., 1983). Currently there is interest in whether this property is requirement for the induction of utologous lymphoprolifertive responses by tumour ntigens (Guerry et l., 1984). The vilbility of McAbs to the frmework determinnts of MHC Clss I nd Clss II products s well s to leucocyte differentition ntigens permits simultneous immunohistochemicl nlysis The Mcmilln Press Ltd., 1985

2 552 A. FERGUSON et l. of the MHC sttus of tumour cells nd the in situ host response in mnner which hs hitherto proved impossible using conventionl histologicl techniques. In this study we extend this pproch, previously pplied in this lbortory to brest nd colorectl crcinoms (Whitewell et l., 1984; Csib et l., 1984) to comprison of neoplsms of the cervix, endometrium nd ovry, with their norml tissue counterprts. Ptients nd methods Ptients Non-neoplstic cervicl nd endometril tissues were obtined from ptients (ged yers) undergoing totl hysterectomy. The normlity of the tissues nd their hormonl sttus were confirmed by histologicl screening. Norml ovrin tissue ws generlly obtined from ptients of menopusl ge undergoing hysterectomy t the time of the menopuse, when bilterl slpingo-oophorectomy is often routinely crried out t the sme time. The indictions for hysterectomy opertions in this group were usully bnorml bleeding or fibroids nd the ges of the ptients rnged from yers. Specimens judged histologiclly to hve pthologicl chnges were discrded. Endometril crcinoms were likewise obtined from hysterectomy specimens nd cervicl neoplsms from Wertheims hysterectomy specimens nd on one occsion by cervicl biopsy. Ptients with cervicl nd endometril crcinom were ged from nd yers respectively. Ovrin tumours were obtined t lprotomy. The ge rnge of these ptients ws 5-69 yers. Histopthologicl dt on ech specimen re incorported into Tbles II, III nd IV. Processing of specimens Tissues were snp frozen within one hour of surgicl removl nd stored over liquid nitrogen. Seril sections (5-1 pm) were cut, mounted on glss slides nd ir-dried. Ech tissue ws smpled t three different plnes, ll three being mounted on the sme slide. When necessry, slides could be stored t -2 C under dessicted conditions for up to one month. Prior to stining, slides were returned to room temperture, fixed in cetone for 5 min, ir-dried nd immersed in 2% new born clf serum in PBS, ph 7.5. Therefter sections were treted ccording to procedures previously described from this lbortory (Whitwell et l., 1984). Briefly, sections were incubted in the monoclonl ntibody (McAb) first lyer, wshed nd then exposed to diluted horse rdish peroxidse-conjugted rbbit nti-mouse Ig (Dko) contining norml humn serum. The peroxidse rection ws developed in diminobenzidine contining freshly dded H22, the sections wshed nd counterstined in Gills No. 2 Hemlum nd mounted. The specificity of the McAbs ws systemticlly checked on sections of pltine tonsil, lymph node or spleen, ginst which new btches of regents were lso titrted. No ttempt ws mde to bolish endogenous stining, which when present, ws redily distinguishble from specific immunostining. Tissue rections were scored semiquntittively (see footnote to Tble II) s previously described (Whitwell et l., 1984). In most instnces, morphometric nlyses were precluded by the intr-tissue vrition cross given section. Monoclonl ntibodies (McAbs) With the exception of the B73.1 regent, detils of the McAbs used in this study hve been given previously (Whitwell et l., 1984; Csib et l., 1984) nd re summrised in Tble I. Results Clinicopthologicl nd immunohistologicl dt on seril sections from norml tissues nd mlignnt tumours re summrised in Tbles II (ovry), III (cervix) nd IV (endometrium) nd illustrtive exmples of specific immunostining re given in the Figures. A feture common to ll tissues exmined ws the pucity of cells of NK phenotype. Only the occsionl B73.1 or Leu 7 (HNK1) cell ws detected in smll minority of some 6 tissues exmined. This ws so in mlignnt tumours despite the often lrge increse in leucocytes over norml tissues. B73. 1 cells were routinely detectble in norml humn spleen nd Leu 7 (HNKI) cells in the germinl centres of lymphoid tissue (posotive controls). Norml ovry MHC Clss I products (rective with 2A1) were invribly detectble on the germinl epithelium, follicle lining cells nd endothelil cells nd with much less stining intensity on the scttered stroml cells. MHC Clss II products (rective with TDR 31.1) were lso detectble on endothelil nd follicle lining cells but bsent from epithelil nd connective tissue cells. Few DR leucocytes were seen. 2D1 leucocytes were evenly but sprsely distributed throughout the ovrin strom. Comprison of UCHT1 stining with tht of

3 MHC PRODUCTS AND LEUCOCYTE ANTIGENS IN GYNAECOLOGICAL TUMOURS 553 Tble I Monoclonl ntibodies (McAbs) to mjor histocomptbility complex (MHC) products nd leucocyte differentition ntigens Ig clssl McAb (murine)b subclss Specificity Origin References 2A1 IgGI nti HLA Clss I (HLA-A, -B, -C) P.C.L. Beverley Beverley, 198. TDR31.1 IgGi nti HLA Clss II (HLA-DR) J. Bodmer DeKretser et l., D1 IgGI Common leucocyte ntigen P.C.L. Beverley Beverley et l., 198 UCHT1 IgGI T cell receptor ssocited molecule P.C.L. Beverley Cllrd et l., 1981 (gp 19,) (This regent possesses identicl rectivity to OKT3). Kung et l., 1979 OKT4 IgG2b T helper/inducer subset Ortho Kung et l., 1979; Reinherz et l., 1979, b OKT8 IgG2 T cytotoxic/suppressor subset Ortho Reinherz et l., 198 OKMI IgG2b C3bi receptor (rective with Ortho Brerd et l., 198 monocytes/mcrophges; lrge grnulr lymphocytes) Leu 7 (HNKl) IgM Lrge grnulr lymphocytes (LGL): C.M. Blch Abo & Blch, 1981; T cell subset Abo et l., MAS2 IgGl B cells Ser Lb. J. Hbeshw' (personl communiction) B73.1 IgGl Lrge grnulr lymphocytes (LGL) G. Trinchieri (Perussi et l., 1983, b) (FcyR) Rective with inter- nd intr-folliculr B cells (determined on pltine tonsils) nd probbly rective with polymorphic B cell determinnt; bsecond lyer regent: Horse rdish peroxidse conjugted rbbit nti-mouse Ig (Dko). MAS2 suggested tht, lthough few, the numbers of T nd B lymphocytes were pproximtely even, with those of the OKT8 subset more frequently detectble thn those of the OKT4 subset. Leucocytes were minly ssocited with blood vessels nd follicles, nd included some OKM 1 cells in qurter of the tissues exmined. In one ovry contining corpus luteum, the lutel cells were stined with OKM 1 nd MAS2. Interestingly, the epithelil cells of one benign tumour ( serous cystdenomen) were positive for both Clss I nd Clss II products. Ovrin crcinom (Tble II) Seven ovrin crcinoms exmined filed to express MHC Clss I products regrdless of histologicl type or clinicl grde but stroml cells expressed the ntigens strongly. However, not ll histologicl ctegories of ovrin neoplsm were represented. Contrriwise, 4/8 crcinoms expressed DR ntigens (Figures 1 nd 2) where none wbre detectble in norml epithelium. Stining ws clerly ssocited with the mlignnt cells where it ws either uniform (Figure 1) or heterogeneous (Figure 2) lthough DR' leucocytes (? B cells, monocytes, ctivted T cells) were lso detected in the strom of mny tumours. Seven tumours were chrcterised by mssive influx of 2D1 leucocytes which ws unrelted to the degree of necrosis. While these were most numerous in the strom, in 6/8 specimens leucocytes hd lso penetrted the tumour mss. Comprison of stining with the vrious McAbs indicted tht the leucocyte strom consisted of T cells in excess of B cells nd monocytes/ mcrophges. OKT4 cells were detectble in 6/8 smples nd OKT8 cells in 8/8. Overll, OKT8 cells obviously exceeded OKT4 cells in 3/8 cses. All tumours contined OKM 1 cells, mostly in the strom, but lso in res of tumour necrosis. Norml cervix MHC Clss I ntigens were strongly expressed on the lower one-third to one hlf of the squmous epithelium (Figure 3). In 1/14 smples endocervicl glnds were present, which were Clss I positive. There ws no obvious correltion with the hormonl sttus of the women. MHC Clss II ntigens were indetectble on epithelil cells but lmost ll tissues reveled few DR leucocytes scttered t the bse of the squmous epithelium nd round the squmocolumnr junction nd endocervicl glnds. Seven of 1 smples which contined endocervicl glnds exhibited Clss II stining of the glndulr cells which ws gin pprently unrelted to the hormonl sttus of the women. 2D1 leucocytes were present in moderte numbers in 13/13 specimens. They were most F

4 554 A. FERGUSON et l. ' Co e. c I.- CO r u ) o cn fa) : o 1- - k (Z k I I I I I I I - -I- - COo So r.-) -r ) So I, -. -~ CO C.~ _ ) W #= O) Q O~Q C* CO 4 IC en Co C O CO E E Cd~~~~~~~~~~~~~~~~~~~C r _) CO CO o~~~~~~~~cw ~~CO )* -* C )~~CO ) _ of - - ))Sd ~ ~ ) ))CZ )' Q Cd z o Cd~~~ Cd CO F CO C ;V CO

5 MHC PRODUCTS AND LEUCOCYTE ANTIGENS IN GYNAECOLOGICAL TUMOURS 555 ''"* Figure 1 Ovrin endometrioid denocrcinom stined for MHC Clss II (TDR 31.1) nd showing strong nd uniform expression on the tumour cells, but not on the supporting strom. Counterstined with hemtoxylin ( x 22). Figure 2 Poorly differentited ovrin denocrcinom stined for MHC Clss II (TDR 31.1) showing heterogeneity of expression mong the tumour cells. The supporting stroml tissue is negtive but contins some DR' leucocytes. Counterstined with hemtoxylin ( x 22). Al. ~ ~ ~ ~.w -, *.4J. Figure 3 Norml cervicl squmous epithelium stined for MHC Clss I (2A1) showing pronounced uniform expression in the lower one-third to one-hlf of the squmous epithelium. Scttered leucocytes nd vessel lining cells in the sub-epithelil region re lso positive. Counterstined with hemtoxylin ( x 22). numerous t the bse of the squmous epithelium t the squmo-columnr junction nd round endocervicl glnds. They were lso found scttered throughout the lower hlf of the squmous epithelium. Most of the 2D1 popultions were lso UCHTI indicting predominnce of T cells (Figure 4) but not to the exclusion of other cell types. OKT8 cells were more consistently demonstrble (12/12 tissues) nd numericlly superior to OKT4 cells (detected in 8/13 tissues). Eleven of 12 tissues contined MAS2 cells, lbeit in low numbers round endocervicl glnds which in most instnces were lso stined with this regent. OKM 1 cells were eqully sprsely represented in 9/14 tissues, with slight excess t the squmo-columnr junction. Figure 4 Norml cervicl squmous epithelium stined for T lymphocytes (UCHTI) showing numerous positive cells t the bse nd in the lower hlf of the squmous epithelium, nd in the underlying connective tissue. Counterstined with hemtoxylin ( x 22). Cervicl crcinom (Tble III) Six of 1 cervicl crcinoms filed to express MHC Clss I ntigens (Figure 5). One (ptient JS, Tble III) exhibited uniform 2A1 stining nd three others (JC, SC, JH) heterogeneous stining. Two crcinoms (ptients JC, SC) reveled DR positivity of the tumour cells. All 1 crcinoms were mssively infiltrted with 2D1 leucocytes which hd predominntly stroml loclistion, though in 8 neoplsms there ws lso significnt penetrtion of the tumour mss. Agin, 2D1 cell infiltrtion ws not relted to tumour necrosis. Comprison with UCHTI stining indicted tht the mjority of 2D1 cells were usully T cells with the OKT8 subset clerly exceeding the OKT4 subset in 5/1 cses. However, B cells (MAS2) were present in the leucocytic

6 556 A. FERGUSON et l. 8 C. C) C.) C. k ~o k I I -I -I- ~~ ~ -L U) v C) cz C) C.) C.).t ey C.) - CU -- Nr (In tn 'I ct. - x, C)~ > ~ =.d - cd (A 8 2 C- cd :j e (. Z.4 *~~~~~~t o Cd CO) U)C (U L C)C)~s C- r *occ cd ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~CU u 4-4 W --~~~~~~~ IE c 4 - C 3 d CA C.)

7 MHC PRODUCTS AND LEUCOCYTE ANTIGENS IN GYNAECOLOGICAL TUMOURS 557 ~~~~r.~~~~v Figure 5 Cervicl crcinom stined for MHC Clss I (2A1) showing bsence from the tumour cells in contrst with positive expression on stroml nd endothelil cells. Counterstined with hemtoxylin (x22). *f A#4. e. Figure 6 Cervicl crcinom stined for monocytes/ mcrophges (OKMI) showing numerous positive cells within the tumour mss ( x 22). strom in 8/1 specimens (in reltively lrge numbers in five of these). In two cses this regent lso stined the tumour cells. Nine of 1 smples contined substntil numbers of OKM 1 cells mostly confined to the strom but sometimes in close juxtposition to tumour cells (Figure 6). Stining with OKM 1 ws lso mrked t the necrotic centre of three neoplsms. Norml endometrium Cells comprising the endometril glnds, myometril tissue, endothelium nd the stroml component of the endometrium were uniformly 2A1 nd in 6/13 cses the endometril glnds were lso Clss II positive. This ltter property ws not relted to the hormonl sttus of the ptients. 2D 1 leucocytes were present in moderte numbers in ll 13 tissues exmined, smll proportion of which were DR. These were more numerous in the endometrium thn in the underlying myometrium nd tended to cluster round the endometril glnds. The leucocytes consisted predominntly of T cells of which OKT8 cells were more consistently detected (12/12 cses) thn OKT4 cells (9/13 cses). Ten of 12 specimens contined low numbers of B cells nd there were lso few OKM 1 cells. Some norml endometril glnds were stined with Leu 7 (HNKI) nd MAS2. There ws little pprent quntittive vrition in the number nd subtype of leucocytes during the vrying phses of the menstrul cycle. Endometril crcinom (Tble IV) Four of 8 tumours were entirely lcking in MHC Clss I ntigen nd expression in 4 others ws heterogeneous. Stroml tissue ws strongly positive in ll cses. There ws no correltion between ntigen expression nd tumour grde. Only 2/8 tumours (ptients BC nd FH) expressed DR ntigens on the epithelil cells (cf. Figure 7); these tumours were well differentited nd the pttern of stining ws heterogeneous. 2D1 cells were present in lrge numbers in ll 8 tumours exmined. These were especilly numerous t the junctionl res between tumour nd norml tissue nd in the tumour strom. Five smples showed significnt penetrtion of the tumour mss. Most of the 2D1 leucocytes were T cells. Agin, in 4/8 smples OKT8 cells were numericlly superior to OKT4 cells. In this series, lymphocytes which hd penetrted the tumour mss were predominntly of OKT8 phenotype (Figure 8) nd Figure 7 Endometril denocrcinom stined for MHC Clss II (TDR 31.1) showing bsence from the tumour cells but positive expression on vessels nd leucocytes within the tumour mss. Also shown is n re of rchitecturl typi djcent to the tumour, the stining pttern of which is heterogeneous. Counterstined with hemtoxylin ( x 22). G

8 558 A. FERGUSON et l. CA r. cd._ E ~ ~~ u) -o 4._ E o oc ~. 3OU I--._ = I - e 'IT o n NT o. CNI C-n U) kr ra Z * t: ~Z c C..C cdc C d ^ E ra~~~~~ i U.- -u C) u 1:1

9 MHC PRODUCTS AND LEUCOCYTE ANTIGENS IN GYNAECOLOGICAL TUMOURS 559 Figure 8 Well differentited denocrcinom of endo- Figure 9 Adenocrcinom of endometrium stined metrium showing hevy ingress of suppressor/ for MHC Clss II (TDR 31.1) showing numerous cytotoxic (OKT8) T cells. Counterstined with DR stroml leucocytes of lymphocyte nd mcrohemtoxylin ( x 22). phge morphology, nd DR- epithelil cells. Counterstined with hemtoxylin ( x 22). DR' leucocytes which comprised mcrophges s well s lymphocytes (Figure 9). B cells were lso represented in moderte numbers in the strom, but were exceeded by cells of OKM1 phenotype, which were lso detectble in the tumour mss (5/8 cses). In 4 of the 8 neoplsms, pprent crossrectivity with MAS2 of the most differentited tumour cells ws in evidence. In ddition, some fibrous septl res between endometril tumour msses were stined with Leu 7 (HNK1). Discussion While severl tumour-ssocited ntigens of gynecologicl neoplsms hve been detected serologiclly (Bst et l., 1983; Bhttchry et l., 1982; Msuho et l., 1984) their bility to evoke immune responses in the utochthonous host is unknown. Accordingly, ttention ws focussed on the MHC products of tumour cells which re likely to be involved in immune induction nd tumour cell recognition nd the nture of the in situ inflmmtory cells s defined by monoclonl ntibodies. In respect of these properties this study hs disclosed number of potentilly importnt differences between mlignnt gynecologicl tumours nd their norml tissue counterprts. It is preliminry to the extent tht the pnel of McAbs ws limited s were the numbers of tumours exmined of ech of the mjor types. Mlignnt epithelil cells were frequently MHC Clss I negtive, under conditions where the strom ws strongly positive. The difference from norml tissue ws most mrked in the cse of cervicl nd ovrin crcinom where the mjority of tutmours were negtive. A trend towrds Clss I negtivity mong endometrioid ovrin neoplsms ws reported by Kbwt et l. (1983). Our filure to encounter Clss I positive tumours in our smller series is thus probbly reflection of the predominnce of these histologicl types rther thn of differentil stining sensitivity. To our knowledge, the HLA Clss I sttus of cervix nd endometril crcinoms nd their norml tissue counterprts hs not been previously reported (cf. Dr et l., 1984). Depressed or heterogeneous expression of MHC Clss I products, s detected by immunohistologicl procedures, is property of some tumours of diverse histogenic derivtion including brest (Fleming et l., 1981; Bhn & Des Mris, 1983; Rowe & Beverley, 1984; Whitwell et l., 1984) nd to lesser extent, colorectl crcinom (Csib et l., 1984). Other neoplsms, notbly mlignnt melnom (Ruiter et l., 1982) nd certin histologicl ctegories of ovrin crcinom (Kbwt et l., 1983) pper to express Clss I ntigens with greter consistency, phenomenon which is pprently relted to the predominntly T cell infiltrte which is feture of these tumours. The level of the filure of primry tumours to express Clss I ntigens hs yet to be elucidted. The immunoperoxidse method s employed in this study is essentilly qulittive technique, so tht levels of Clss I expression below the threshold limits of detection, could conceivbly occur, i.e. the deficit my be reltive, rther thn bsolute. As exemplified by genetic experiments with Dudi cells (Arce-Gomez et l., 1978) some tumours my fil to express HLA due to lck of /2 microglobulin (/2 i), the low moleculr weight glycoprotein required to stblize the Clss I hevy chin.

10 56 A. FERGUSON et l. The extent to which the gynecologicl neoplsms in this study synthesised nd secreted P2 m ws not scertined. Filure to express MHC Clss I ntigens by mlignnt cells rising from HLApositive tissues could, s Bodmer (1981) hs dvocted, be chnge tht could be selected for during tumour progression through the dvntge of resistnce to ttck by cytolytic T cells. Such resistnce might presumbly only be n dvntge to tumours tht express tumour-ssocited ntigens cpble of evoking T cell immunity. However, this hypothesis my now require some revision since, t the clonl level, T cells lcking NK-like ctivity my recognise certin tumour types in n immunologiclly non-restricted fshion (De Vries & Spits, 1984). Thus, it my not be without significnce in our study tht OKT8 cell influx ws not correlted with HLA Clss I sttus. It is lso possible tht lck of Clss I determinnts influences cell:cell interctions outside the immune system in wy, e.g. tht fvours the emergence of metsttic vrints. In common with other tissues, MHC Clss II molecules, normlly considered to be confined to cells of the immune system were frequently detectble on the glndulr epitheli of norml cervix nd endometrium (cf. Dr et l., 1984b). The so-clled 'berrnt expression' of Clss II products on norml epithelil cells is ssocited with immunologicl, inflmmtory s well s hormonl stimuli (Klreskog et l., 198; Lmpert et l., 1981; Mson et l., 1981; Selby et l., 1983) nd similr stimuli could promote their expression on mlignnt epithelil cells. In cervicl nd endometril tumours MHC Clss II expression ppers to reflect the cellulr origin of the neoplsm. On the other hnd, tumours originting from pprently DR-negtive epithelium (ovry) could cquire the bility to express Clss II molecules in response to stimuli of the type mentioned bove. In both utoimmune nd neoplstic diseses MHC Clss II products on trget cells re envisged s vehicle for the presenttion of utontigen to T helper/inducer lymphocytes on the ssumption tht the configurtion of the prticulr Clss II product fits tht of the utontigen (Londei et l., 1984). In mlignnt melnom, for instnce, utologous lymphoprolifertive responses cn be induced by DR-positive tumour cells (Guerry et l., 1984) but the extent to which similr responses re evoked ginst other ctegories of humn mlignncy hs yet to be determined. In our series, the four DR' ovrin tumours ppered to show no excess T4 influx. Anlysis of the inflmmtory infiltrtes with McAbs to leucocyte differentition ntigens confirmed tht infiltrtion occurs, to very mrked extent, in mny gynecologicl tumours. All ctegories of leucocyte rective with McAbs in this series were incresed in tumours in comprison with norml tissues. The gretest contrst ws in crcinom of the ovry, minly becuse in the norml tissue leucocytes were few. In norml cervix where moderte numbers of leucocytes were consistently detectble nd were mostly T cells, the gretest diference between norml nd tumour tissues ws the incresed number of B cells nd OKM I' mcrophges. B cells were likewise present in moderte numbers in ovrin nd endometril crcinoms but not s numerous s in cervicl crcinom, where their numbers sometimes pproched those of T cells. The extent to which these hd differentited into plsm cells ws not esily discernible on cryostt sections. Since OKM 1 cells could not lwys be unequivoclly identified s mcrophges by morphologicl criteri, nd 8% of the lrge grnulr lymphocyte (LGL) subset of peripherl blood is OKM1 (Ortldo et l., 1981), the LGL popultion ws monitored in sections with two McAbs, one of which (B73.1) is rective with virtully the entire LGL popultion (Perussi et l., 1983,b) nd nother (Leu 7; HNK1) which rects with pproximtely 75% of LGL s well s subset of T (suppressor) cells (Abo & Blch, 1981; Abo et l., 1982,b). B73.1 or Leu 7 cells were scrcely ever seen, indicting tht the OKM1 popultion ws probbly wholly of monocyte/mcrophge composition. In ovrin cncer, there is mrked size vrition nd cytochemicl heterogeneity mong mcrophges which is ssocited with spectrum of ctivities from suppression to ntibody-dependent cellulr cytotoxicity (Hskill et l., 1982b). However, gynecologicl cncers, like those of brest, colon nd lung (Wtnbe et l., 1983; Bhn & Des Mris, 1983; Whitwell et l., 1984; Csib et l., 1984), contin few cells of nturl killer phenotype. This conclusion is in concordnce with erlier functionl nd morphologicl dt on vriety of tumours from severl lbortories (Moore & Vose, 1981; Eremin et l., 1981; Pizzolo et l., 1984) nd on ovrin cncer, in prticulr (Intron et l., 1983; Kbwt et l., 1983). The implictions re tht NK cells, which re mximlly expressed in blood nd spleen rrely extrvste nd ny direct nti-tumour ctivity is consequently very limited or non-existent. The stimuli to leucocyte ingress into solid neoplsms re still lrgely unknown though the presumption remins tht tumour ntigenicity is mjor, but certinly not the only fctor. Whether humn gynecologicl neoplsms re immunogenic

11 MHC PRODUCTS AND LEUCOCYTE ANTIGENS IN GYNAECOLOGICAL TUMOURS 561 in the utologous host or not, the demonstrtion tht the extrvstion of leucocytes is not rndom event might be relevnt to host reponses of n immune nture. Although niopliometric nlysis of infiltrting popultions is often complicted by mrked heterogeneity in given section, it is cler tht some popultions predominte over others. In mny sections, there ws little doubt tht T8 cells exceeded T4 cells, indictive of shift from the proportions normlly present in peripherl blood, though perhps to lesser extent from those in ptients with progressive or dvnced disese (cf. McCluskey et l., 1983). Similrly s lredy noted, in cervicl crcinom there ws significnt ingress of B cells which ws not consistent with rndom extrvstion. However, it is possible tht in certin crcinoms (e.g. endometril), the ssocition of T8 lymphocytes with epithelil cells is not indictive of rection to the tumour t ll, but is rther reflection of the norml reltionship between the lymphocytes nd epithelil cells in this tissue. Functionl studies showing depressed prolifertive ctivity in T cells recovered from ovrin cncers implicted tumour inctivtion s the cuse, or filure of prticulr subset to loclise t the tumour site (Hskill et l., 1982). An lterntive explntion might be tht the predominnt T8 subset contins functionlly ctive suppressor cells (Vose & Moore, 1979). This possibility, which would theoreticlly fvour tumour growth, might not be entirely unexpected in tumours which, prt from therpeutic intervention, hve 'escped' beyond recll s would doubtless hve been the cse for the tumours in this study. At this point, lte in the progression of the lesions, the mgnitude nd type of cellulr immune response is likely to hve little in vivo opertionl significnce to the dvntge of the host. This study ws supported by grnts from the Cncer Reserch Cmpign of Gret Britin. We thnk Dr Anne Simmonds, Glsgow Royl Infirmry, for contributing dditionl tissue to this study. References ABO, T. & BALCH, C.M. (1981). A differentition ntigen of humn NK nd K cells identified by monoclonl ntibody (HNK-1). J. Immunol., 127, 124. ABO, T., COOPER, M.D. & BALCH, C.M. (1982). Chrcteristion of HNK-1 () (Leu-7) humn lymphocytes. I. Two distinct phenotypes of humn NK cells with different cytotoxic cpbility. J. Immunol., 129, ABO, T., COOPER, M.D. & BALCH, C.M. (1982b). Postntl expnsion of the nturl killer nd killer cell popultion in humns identified by the monoclonl HNK-1 ntibody. J. Exp. Med., 155, 321. ARCE-GOMEZ, B., JONES, E.A., BARNSTABLE, C.J., SOLOMON, E. & BODMER, W.F. (1978). The genetic control of HLA-A nd B ntigens in somtic cell hybrids: requirement for 2 microglobulin. Tissue Antigens, 11, 112. BAST, R.C., KLUG, T.L., ST. JOHN, E. & 9 others (1983). A rdioimmunossy using monoclonl ntibody to monitor the course of epithelil ovrin cncer. N. Engl. J. Med., 38, 883. BHATTACHARYA, M., CHATTERJEE, S.K., BARLOW, J.J. & FUJI, H. (1982). Monoclonl ntibodies recognising tumour-ssocited ntigens of humn ovrin mucinous cysdenocrcinoms. Cncer Res., 42, 165. BEVERLEY, P.C.L. (198). Production nd use of monoclonl ntibodies in trnsplnttion immunology. In Trnsplnttion nd Clinicl Immunology XI Tourine et l. (eds) p.87. Excerpt Medic: Amsterdm. BEVERLEY, P., LINCH, D. & DELIA, D. (198). Isoltion of humn hemtopoietic progenitor cells using monoclonl ntibodies. Nture, 287, 332. BHAN, A.K. & DES MARAIS, E.L. (1983). Immunohistologic chrcteristion of mjor histocomptibility ntigens nd inflmmtory cellulr infiltrte in humn brest cncer. J. Ntl Cncer Inst., 71, 57. BODMER, W.F. (1981). HLA structure nd function: A contemporry view. Tissue Antigens, 17, 9. BREARD, J., REINHERZ, E.L., KUNG, P.C., GOLDSTEIN, G. & SCHLOSSMAN, S.F. (198). A monoclonl ntibody rective with humn peripherl blood monocytes. J. Immunol., 124, CALLARD, R.E., SMITH, C.M., WOMAN, C., LINCH, D., CAWLEY, J.C. & BEVERLEY, P.C.L. (1981). Unusul phenotype nd function of n expnded subpopultion of T cells in ptients with hemtopoietic disorders. Clin. Exp. Immunol., 43, 497. CSIBA, A., WHITEWELL, H.L. & MOORE, M. (1984). Distribution of histocomptibility nd leucocyte differentition ntigens in norml humn colon nd in benign nd mlignnt colonic neoplsms. Br. J. Cncer, 5, 699. DAAR, A.S. & FABRE, J.W. (1983). The membrne ntigens of humn colorectl cncer cells: Demonstrtion with monoclonl ntibodies of heterogeneity within nd between tumours nd of nomlous expression of HLA-DR. Eur. J. Cncer Clin. Oncol., 19, 29. DAAR, A.S., FUGGLE, S.V., FABRE, J.W., TING, A. & MORRIS, P.J. (1984). The detiled distribution of HLA-A,B,C ntigens in norml humn orgns. Trnsplnttion, 38, 287.

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