Identification of Subpopulations of Mononuclear Cells in Cutaneous Infiltrates I. Differentiation Between B Cells, T Cells, and Histiocytes

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IDE~TIFICATIO:\ OF

SUBPOPL LATIO:\S OF MONO:\LICLEAR CELLS ClJTA:\EOl S INFILTRATES I. Dt~'FERE1\1'1Afl01\ BET\\EE' B C'EI.I.'i, T CEu.~. ·\l'oOD H tsTronn:s

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J\IICIIAEL M fHA'\K

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<:HEE'. J\1 D ABSTRACT Otlleremiation bE;>t\\l'tm bone marrO\\ clt'rt\NI hmphocyles 1H relbt, lhymu,.. dert\ed I) mphorvtes !T l'Pib). and h tstion le,., has hren arhtPved in t is!-U!' ,.,ert ton,.. through ut iiL~.ation of knol\ n dif!erencl's in cl'll memlmtnt• recepton;. Sheep erylhrocytt·~ (EJ Wl're coated with en her lgGor lgl\.1 antihod) (Alw lorm lgG or lgM EA. lg:\1 EA was mcubmrd w11 h mou,..e corn plement ICl to form lg\1 EAC ('ryo,..ect inn~ w!'re layered with lgl\1 EAC'. lg::\1 EA. or lg(; EA. nnd suhpopulattons ol mononuclear cells t·nuld be dt!'.llll!{Ut>'hed from onr another lw the »dherrnce or nonadhl•rem·e of these reagent,.. H cells bound lg::\1 EAC. hut not lgM EA or lgG EA; histiocytes bound li{M EAC and lgG EA. b Jt not lg:\1 EA; and T rdl~ failed to hind an~ ul lhe,..e reag-enb. H cells were tdenttli('{l tn a curaneous leukemic mliltrnte tn a pattent with a H cell leukemta. H ist ioc~ t es \\ert• idl'nt ified mfil t rating the dermis and t•piderm ·~ m a graft versu,..ho:,.t reaction. A dermall)mphocyrtc tnf'iltrale tn a squamou~ <"I'll tarcinoma lesion did not bind lgG EA or lgl\1 E:\C. The remaindrr of 1he fresh tis:,.ue from thb le:-wn was finely mtnced and ftltered lhrough a f!lass wool column. The 1\·mphoc:o-le,.. in the filtrate \\l're pusiti,·e for human thymic lymphocyte anttgcn. rstnhlishing their T celltdenttty. It '" 1here fore pos,.,ible to di,..tinguish bet wet·n H cells. T celb, and ht,..t tocvle:- tn "ktn Je,.,wns. This method ,.,hould be valuable tn thl' unestigatton uf cutaneous mononudear infiltrale~ through identilicntton of effector cells In recent years it has bet·nme rlrar that

l~·m­

the chtcken the~ develt)p under the tnfluem·t• of the hursa of Fabricius but 111 humans mil\ pa~s di· and lunrtionally distinct groups. The firs1 !n'Ollp recti\ !rom the hont> marrow to peripheral lymphundergoes final different tal ton under 1 he inlluerwe md org-ans [6 j. B cells hear easil) dNectahle of the thymus and rs rnmpri"l'O of th~ mus-d<'m Pd ~urfare immunoglobulin F) and undergo blast lymphocvte,.. IT cells). T cells play a central rolt• tn 1ransformat wn on exposure to endotoxrn [" ). grah reJeCt ton and dela~ ed hypersen::.il i' i1y II J 1\ormal lvmphoid tissue i~ organtzed rnto thyand collaborate ,\ith the precursors of antibody- mic-dependent areas where T celb predommate, forming r<'lls in produclton ol the humoral rl'- and thymic-independent arens. where H tells presponse 10 heterologous ery1hror~te~. ::.erum pro- domtnale [9]. The majority of thl' cell~ m 1he teins, and hapten -protein conJugale" 1~. ;~ j. T cell~ dtffu~e cortex oil\ mph node~ and the peria rlertn· do not hear ea:.ily detertablt• tmmunoglobulm Ill lar area ot1he whttE;> pulp in spl~>ens are T cells, and and undergo hlast t ranslormatwn "hen exposed to H cells comprtse the maJority ol cell,.. in the the plant mitogen~ phytohema~-:glutmin and ron· prtman Jollrcle,.. and germmal renter!oo. canavnlin A [.)].The second group is comprised of Thr t hrrd relltypr re!'ol{"nized to be i mportanl in the bone marrow·derived lvmphocytes tH rellsl. the gene~is or both cellular and humoraltmmuntty the prt>nrrsors of the an1ihod) -producing cell:-;. In brlongs to the pha!-(<>l'yle ,.,eries and include;. histiorvtes and mononte,.. Hv;,tander celb of this gr<.>up become at·tivated b~ mediator::. relenscd lmm stimulated lvmphocytes and are tmportant Manu,nipt n•rt>tn•d \1.tn·h II!. 197'l, 111 revt"l'd lnrm effector cells in rellulnr t m mune react tons po. 11]. April :1. J!l';:l: !l('('l'Ptl'd lor puhhl'alton .\pril l!l. 1!1';:1. I'he,.,e three tvpe" ot mononuclear cells l'lln he Prt'sl•ntt>d tn p11rt at thf• .Joint '\at tunal \leettnt.:~ ol 1ht• Amennm FE"dt•rallon lor l'ltnK.tl f{e,eurch nnd tht· distinguished from nne .mother berausr of dtllerAmeril·nn Sociel\ lnr Chnil·ul lnn-,t igattnn in Atlantic l'nces tn membrane receptors and dillerenttatton l'tty 'l'"' .)l'r'l'~. April ~~1. Hli:l. From the Dl'rmatolngyt and lmmunnlngyt Branche, anltgens. B l'ells [I :Z J and cells of I he phagoc~ te nf the 'atiunal C'ancl'r ln>lllut<• and lhl' Laburutnries ctl sertes [13] bear rereptor!oo lor the actinlled third Chmcallme~tigatton* and Jmmunnlnl(yl of the 'alwnal component of complement (C:l). In some species lnMIIUtl' nl Allerg~ and lnleclious Ot~l'ase. l\nltnnal the C:l receptor ol monocytes. but not of H cells. l n~titutes uf Health. Bethe~da. Ma~land. tRepnnt re requires Mg• for 1ts lunrtion. For the human que~t~ to: Dr R. L Edt>lsnn. Hnom 1:''\~:lx. Budding to. :--Jutwnul lnsttlutl's ol lle.1hh Ht.>the,..da. Maryland monoc\te (':~ receptor there i!i no such require:l0011.l ment. ·and these ('l'lls must be distingutshed !rom phocytc~ ran he di' ided tnto l wu ont ogl'nl'ticall\

MONO:-..LTLEAH CELLS IN CTTANF.Ol 'S 1!\Fil.TRATES

one another hy the pre;.ence of II(G recepton. on cell;. of the phagon t e ~ene,.. hut not on B cell~ [ 1 I, 111 ]. Human T cells bear neither a C:l nor lgG receptor but under appropriate expenmental conditions form nonimmune rosettes with sheep erythrocyte;. j16. li ]. In mtre 11 I and humans [1~)], T cells ran also he identi!ied by the presence of specifi(' dtfferentiatwn antigen,. It has been posst ble to locale H cell area" in ussue sections of normal mou;.e [:lO I and human [2l]lymphoid tissue through utilization o l C:~ rece pto rs, and it ha~:. been reported that T cell areas could he identified in normal human lymphoid t isf\ue 1h mugh the T cell re<'eptor for shN•p e rythrocyte;. ]21]. The result~ reported here. from the study of three dermatologir diseases, demon st rate the identtficat ion of H cell~ and histtorvtc~ in t is~uc sect wn. a~ "ell as the ident ifirat ion of T cell!i alter :-.eparattnn of the cell,.. from the le,;ton into suspcn:-.ion.

Preparotwn

11; EA

and J::,.\ C

Tht• tvpe nl monunud ..ar cPils tn thE' tb,ul:'~ "tud1ed wa!\ clet fi'Ct rd h\ "'erl;t\ 1111: ~el linn:- wIth ,heep eryt hm· cytp, (El contatntnl(, on thetr hUrfacp,, moietie~ which would Interact wnh th(• speciiH· rect>pl on the "urlau·~ of mnnonude:tr rl"lk E were fi~t c·uutcd with either lgM or lgG antihocl~ tAl tn lnrm l~tM nr li(G EA. I~:G EA w11l adhen· tn thosP c·ells heunn~: a rPceptor tnr lgG imrnunuglubultn. while lJ;I\1 EA w11l not 11(:\1 EA "econdarily coatPd with 1he Ol'tiVIlled thtrd t'nm ponent of c·omplt•· ment ((') to form lgM EA<' \\Ill adhere to thn~c l'E'Ils beunnl( n rN·<•plor lor C:t B cells. T c·rlk and hi,tiOl\tE's can be di,ungtu~ht•d from nnl' annth('r h~ the adherem·e or nonadhPrl:'ncr ul t he"l relllo!<'lll" 1Tuhl" 1 !Whhit anti :-.hl'ep ('ryrhroc\te antibod1e" "ere preparte! w Zealand "hill' rahhns Wl'rr·lmmtm.n·rl with butled , heep erythrot\'le -..twma. the n•,uh1ng h11!h allinity antthodtl'~ \\l're separatrd rrlln lg\1 ;mrl li(G trarunn" hy g!'l fih rat inn un Sl'phadl'x-:!Oo rolumns ancl •ucro-.1' den,.lt\ l(radienl ultrat·rntntugatlnn : and runt·entnnwn" nl anubody usPct to coat the ,.he!'p l'~thmc·ytp,, were chu,en ln prorlure optimal hemo)ysi~ Ul ;r; o ('in I hi.' presenc·p of J!liiOea p1g complPment \1ou"e ~rrum sened a~ the ~oun·p nf complemPnt to lorm the• EAC cnmplexe~ smre mouse rnmplement i, poor!\' lytll Tn lorm EAC. mouse serum was diluted I Ill m vl'ronal buffered saline cunliuning optimal torK!'nt rat 11m" nf ealr1um ,tncl magnp,.1um in 0.1 , J!elatm and wa~ 1hen incubated wnh an equal volume of lgCi or lg\11 EA 11 · JO•/ ml 111 pho~phnte buflerPd snlmel lnr :!0 min at :!7 C. The resulting EAC were washed IWII'l' and re,uspPnclE'd in phosphate buffered hilline tu thr "anw c·ont·entration J'AHLE R('n·ptor., on monmwclrar C('/1,,

Fmun S('c l/lm Trt'olmcnt u·tth EA and EAC

:-iix nunon ~pc·tton~ \\PrP cut with an lnterntltional Harrh 1 r.-c"tal. allow eel to cln at room tPmpNat urP em ~:Ia" ~lid.l·~. and ht\Prl.'cl \\ithlg(; EA. lg:\1 EA. or ll(:\1 E.\(' .\Iter ·o nun at room temperature. the ~hde~ ''erP W-hcd in pho~phatc hufiPred ~aline until all nunadher· l'nt rt'cl l't·ll~ hucl h!'en remm·ed. The~· were t hPn lixPd an eithrr .! glutnralch·h\dt• or Perfix tApplil•d Hw,<·wnc·t. Pallrr,nn. \ .J l for :Jn rn1n and 'lamed wnh hematoxylin .tnd eo~lll

E Tnt

In

1'1.-.,u(•

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Sht•l.'p en 1 hrn<'\ 1e~ werr ~lured at -1 • C 111 Al ~e' N's soluunn unci W('rt• userl within I days after remm·al from thr shcl.'p Bt•fon• usr t ht>y werE' wa~herl tw1re in Eaglr'~ med1um and aclJu'tPd tn iir. 1'1. or ;.,r, v/ v c·nn<·entr;t tions in phosphutt• hutft>rerl saltne. med1um 199 or mc•d1um 11'10 tall nwd1a lrnm the :-;111 l\ll•diu l 1mtl. :\ledw Hill nnd lh10 were selerterl hecause tht•.\ nmtam Glass :.!ide:; containing 6-11 frn1en and ('u· !\le: ~ec·ttmh wrrl' luwn·rl wtt h E a•td incubated at 'l7 C lur I hr 111 a humidit\ l'hamher tn prevent evapmatum. Tht>y wrrr t ht>n tnnrl)ated at 11 ( htr 2 hr ur uvernl!(ht wusht•d in th<· ~llml' merlium a" that tn whlt'h theE had been origi1wll~ ~ust>ended. and lixt•d and -.tamed m the same way as the EA and EAC t remed ~ecllon~ .

f: llrl,l'/lt• f urmullfm

11

Su.,pc•n,wn

Thl' t·'\c'hl.'cl squ.tmnu" rE'll carcinoma 1from patiPIIl dP~c·rihed 111 Ht•,ulhl was !tnPI~ minn•d wnh sr1ssors and tnrcl'ps tnlo Eagle~ medium t1mtatning Ill'' fetal huvtne "erum . ThP mtm·E'd tls,.ue and ~upernatant l'raruon~ wNe llltert•d through p,•rex wool in order w remove unm1m·pd u~suc ancl lar~:e cp1theltal nils. RHC were lv~ed w1th ammonium rhloride. The remaintng lvmphocytrs were \\3-..hPd l\\ll limes in medium und adju~ted lll :! ;) 10'/ml tn nwcl1urn cnnta1ning Ill', fetal ho\'lne serum 100 11lnli1l :t \1\-..u~pcnsiunniF.pllhiOOI'Iofh·mphoc~te miJ
()iT Cf'l/.' b\ (\tutoXH'II\ 7'f•.,lmj.:

Antht•rum "Jll'l'lltcall~ tmllc·to human th~ mucne~ was as prt·vlnusl~ clescribecl 1191. Briefly. rahh1t" were 1mmulllzed with human fetal thymus celb. and the re~ulun~: ~era Wl'rl' absorbed with a mixturE' of cultured human hom· m.~rrow-dPri\'l'cl h mphtlt.\ tPs to remove antthodll'' to 1:3 cells. :-:u~pPnrlpd lvmphun·tp~ ohtamed from the ~quamou~ c·ell cardnoma as dt•s<•ribed abo\'e werP assavecl fur humnn thYilllt' lymphor\lC untigen ! HTLAJ. For HTLA te~t ing 1he l1lt rut!.' was platrd in a minutest plate: 1111 uf anllsl'rlllll wa" added to l.'llt'h well: the plates wt•re mcuhat cd at :11 (' lor I hr and nnsed w remo,·e unbound antihod~ a nd anticomplemt·ntary facton-. One 11l nf ahsorht.'rl rahh11 ~!'rum lc·nmplemE'nt suurt·el was acldecl to em·h wt•ll and tlwn. fulluwl"ll! :!-hr mcuhauun ill runm tl'mJ>PraturP. tr.pan hlul' dyP wa" aclded Cell dPath wa~ determ ined h' stainin~: with II') pan blue dye. the percentage nl ktlled cell" tall ulatcd !rum. prodt~t·cd

' slutned in ahst>nce ul anttsPrum 1111)

', stili ned in ah~ence ol anuserum

100

84

T HE JOURNAL OF t '\\ ESTIGATI \E UERM ATO LO(;Y

Backl(round t·ytntoxititv wa,. pe rcent

con~istcntly le~~

than 10

RFSt LTS

Localization of B Ce/1.,, T Cell.~. and Hi.,twcytc.' Normal L_, mphord Tissue

1n

Because of the known compartmentalization of nor mal lym ph oid tissue into T cell. B cell, and h i~tiocyte areas 19], hi~tologieally normal human lymph nodes a nd spleens. obtained su rgically, were used as controls to demonstrate t he capability of ou r m ethod to iden t ify the::-e t hree cell types in

tis:,ue :-eel ion Crvostat ~ections of t he!>e tissue~ were layered with lgM EA. Igl\1 EAC, or lgG I::A. washed. fixed. and ~tained with Hand E in parallel wtth pathologic specimen~. dE'~cribed sub-,equently . A., ~hown in F'tgure:-. I and 2. the lgt\1 EAC adhered strongly to the follicular areas which are known to be compo.,ed mainly of B cells, with relati,·e sparing of the t h) mus-dependent (T cell) areas. In contrast. l gG EA bind to the tissue hi~tioryte::- (Figure :l), but not to the T and B cell a reas in the splenic white pulp. lgM EA contains neither l he C:l required for adherence to B cells and

Ftc;. I. Sel·tion of normal lymph node treated with lgM J::AC' and \IC\~ecl with dnrk field conden~er. The adherent n~ hrtl(ht ~pot!; o~:ain~t a dark background of mnnonudt•ltr l·elb The EAC adhere to thl' B cells in t\\o follicle~ (arrow~:>). htghlighung the~e areas. The llliPrfollirular rr ct'lll area IS relatively !
EAC appear

Ftc;. 2. Sel'lion of normal ,;pleen treated with IgM EAC T cells ( EAC -negall\e) surround the l'Pntral artenole (straight arrow) "hile t hP ecrentncally placed lolltcle ll'Urwd ltrro\\ l strnngl) hinds EAC becau~e oft he large number of localized B cells. Hematoxvlin and eosin stain. • 100.

8.1

\10 O:-..L' CLEAR CEI.l.!-. IN Cl l'Al\EOU:i 1'\FILTHATF:S

Ftt. ·1. :-.et·uon ot normal spleen treatf'cl Y.ith lgG EA ancl \"lt'\\etl with dark rield conclcn~cr The ttssuf' hts!IOC\'te>of the rcd pulp stronglv hind thi!< reagent. while the Hand T cell nreas in tht> white pulp farrow! are largely negati\'C ~

100.

histiocytes nor lgG h>r which hist iocytes ha\e receptors . Therefore. a.; expected, lg:\1 EA doe~ not adhere to any areas in the section~ or normal tissue. In the studies of pathologic material lgM EA was always used as a contwl against nonspecific binding of sheep erythrocvtes. and in no case wa~ binding with it observed. Thus. areas containing B cells strongly bind lgM EAC. but not lg(; EA: area~ comprised of hi~tt ­ ocytes hind lgG EA and often lg:\1 EAC' . T <·ells bind nett her ot the~e real-{ent~. Se\'eral attempt:; to detect spectfk E cell htndmj.{ toT cells in sections of normal lymph nodes were unsuccessful.

ldenllfwatwn of Leurrmtc B Cells Lymph Node

tn •

'ktn and

A pat tent \dt h t·hronic lymphocytic leukemia presented wtt h a \\'R(' of 671.000/cu m m (91 , lym phncytes I. gerwrali:t.ed lymphadenopathy. and two malar ulcerattons. Previouf, studies demon strated that the penpheral blood lymphocyte~ stained with a polyvalent fluorescein-labeled antihuman tmmunoglobulin reagent. indicating that the abnormal cells were H cells. Punch biopsies from the horders nt the ulcerations shm\ed den!.e focallymphocyw· tnfilt rutef; . F'ungal and bacterial cultures of these ulcerations failed to reveal patho gens. and the lesion~ were considered probable leukemrc infiltrates. The'ie cutaneous lestons and enlarged abdominal lymph node,., from the same pattent tin which normal architecture was replaced with leukemic

cells) offered an opportunity to attempt to identify H celb in lesions ''here they would be expected to be present. t'\earl~ all cells in the lymph nodes (Fig. ll and the majority of cells in the cutaneous 6) could be characterized asH infiltrates (Figs. celb through adherence of IgM EA(' but not of lgG EA.

n.

Tdentificatwn of Infiltrating Histiocvtes /anrou., /,e., wn

ll1

a Cu-

Three weeks following a bone marrow transplam a patient with acute lymphocytic leukemia developed a gralt-versu~-host CG\'HJ reaction characterized hy a generalized exfoliative erythroderma. progressive hepatocellular de!ltruction with markedly elevated transaminases. and melena. Histologic ~ections from involved skin on days Z and 16 of the eruption revealed vacuolar degeneration of ba;,al cells. spongim.is. dyskeratosis. exocytosis of mononucleor cells. and a mononuclear cell infiltrate in the upper derm!s. These findings are consistent with G\' H [2:3). When cryostat hections of skin biopsies, also taken from involved skin on days 2 and 16 of the eruption, were layered with lgG EA and lgM EAC. the ma.iortty of the infiltrating cells were positive with both reagent!> and could be identified as histiocytes ( Ftg. 1). Therefore. it was possible to identify the predommant intiltraung cell in this G\'H reaction as histiocytic. on the basis of cell membrane receptors .

86

THE .JOURNAL OF 1:\VESTI \.ATI\'r: DF:RMATOLOG'

Fu .. 1 lg.\1 ~;. \("treated '<'rtion of hmph node from patil'nt wHh chronic l\'lnphocvttc leukemia. The normal nodal nrl'hitert un· i~ chsruptl'd !)~ the intiltratin~ malignant H cells. :\e.~rl~ all the lymphoc·ytes 111 the section bmd EA(, re.. ulting in a ~ht>et nf EAC which ohscur~ the undt>rlying lymphnntl•s Stained \\'llh hematox~lin and eosin . • 2.10.

F'1c: fi. 1~:.\1 Et\<' lrrutccl st•c·tton nf t•utanenus leukenm· mf'iltrate \'iewed \\llh dark field condenser. The EAC are htghhghted a~ I ight l'tr!'ll•s (Rrro" 1 n.:ainsl mdtst met hnckgrnund ol in tilt rnt ing ct>lls .md cnnnect ive ll"lll' The EAC ~!lO . adhert to the inflltralln!! n·ll~. as expec·11•rl in a R cell infiltrate.

Identijtc·atwn of T Crl/.' in a Cutancou.\ Le.,wn Since we have been unsuccessful in allempts to identify T cells m sect ion h)· use ut the E rosette method. the presence of T cells could be inferred onh by the failure of some cells to hind lgG EA or 11{!\1 EAC . How~\er. the a\·ailabiltty of a serum specifically rytoto\ic fi1r T l'elb allowed us to

rorrohorale the ' I cell tdenllly of inhltratmg cells by line!:-· mincing the fresh tu;;sue and :-.tudying the extracted lymphocytes in su~pension. Pum h biopsy of a Bcmen ·~ mtraepidermal squamous rei I carcinoma in a 6.'l-vear-old male revealed a band-like mononuclear infiltrate in the papillary dermts Tht> lesion. measuring :2 em in diameter, was exrised and di,·ided into two parts: a small

~10 NO:-.:L' C LEAH

CELLS 1:-J Cl TANF.Ol"S li"Fl i.TRATES

portion of the tissue was used for tissue sect ion stu dy: a nd t he majority was fi nely minced a nd fiiLered. as previou!'ly described, to obtain ~uspen ­ s ions of numerous infiltrating rei b. C ryostat sections of the squamouR rei I carci noma failed to bind lgl\1 F:AC' or lgG EA. s uggesting that the inlil t rating cells were T eel b. The ly mphocytes brought in to su~ p en sio n were examined for E recept ors and human thymic ly mphocyte a nt igen (HTLA). :--Jinety-onc percent of 200 counted lymphocytes in the suspen:
R7

formed E ro~ette!-.. Similarly. grrater than 99 percent of the suspended cells we re HTLA positi\ e. Therefore, thl' studies of th e cells in suspension corrohn rated the tissue section s t udies nn d indicatPd that a high percentage of the infilt rating cl.'lls were T cells . OJS(TSSIO'

T he results demonstrate that ou r met hod can distinguish hNween B .:ells. T cells, a nd hts ti orytes in skin lesions through utilization of known

Far. . ti. lgM EA t reaard set·uon ot rutaneous leukemH· inhll rate. Nnnon~J>et·it'it- adherence ot rE'agt•nt to in lilt rating cells is noterl . Hemntoxvhn onrl eosin slain . 2-'>0.

Far.. '; . lgC: EA -treAted section of ~kin fm m patient with l{rafl-ven.us -host reaction . The majunty of I he In fil trating mononuclear cell~ in th i~ lesion a re strongly posit1ve "-ilh thi' rl!agent and are irlenlified a~ histiucytes. One such cell which is infiltrating the epidermis i!o !!hown above the arrow. Hemawxylin and eosin stain. ~ 250.

88

TilE JOURNAL OF 1:--l\ ESTtGATI\ E DF:RMATOLOG\

differences in the s urface st ructu re of these cclb. the availability of a het ero l o!{ou~ serum comaining The three exampl es presen ted were selel"led to antibody s pecifically directed again~! human illustrate the a ppl ica bility of the technique. thymic lymph ocy te antigen [l9) may allow posiConsidering the already tdentllied fun ctiona l tive identihcation of T cells th rough indirect imdifferences between B cells. T cells. and histi - m unoll uore:.cence. ocyte;;, the ty pe of cells pre~ent in lesions an d The diversity of cutaneou~ mononudear in!iltheir distribution <'a n be expected to relate to th e trates and the accesstb il ity ofsk m fo r study shoul d nature of the pathologic proce::;ses. Peripheral make this tissue particularly attractive to those blood leukocytes in c hronic lymphocytic leuke mia investigating the functional roles of lymphocytes pa ti e nts ha,·e been previously charac te rized a, H and histiocytes in pathologic proce:.se~. Sy!-.te ma ti c cells by C~ receptors [24, 25) and surface immu- investigauon:. of the cell population~ pre~ent in a noglobulin [26). and work in progress .- suggests wide ,·a riet\" of cutaneou:. infiltrate, are now in that the va ri ou~ lymphomas can be :-i milarl)< progress . characterized. Recently it has been reported by The uut hon; arf' graH•tul to Or. Murnn Lut 111er tor his Broome, Zucker- F'ranklin , Weiner. Bianco, a nd helpful sugJ!E',t ion,; durin~-: the preparation of the manu Nussenzweig [27) tha t ('i rculattng Sezary cells a re script. to Dr Elame .Jane tor her aid m mterprettng the T cell~. and s tudte" in ou r laboratory suggest a T h1qolol(il' sl'l"IIOns ol lymphmd tissuE'. and to Mr.- Eilt>en cell ongin for the majority of the infiltrating cells Sussman for her teehnicill assistance. in mycosis fungoides. Therefore. it is al ready apparent that certain cutaneoul:l ly mphomatous REH:RF.l\CES and leu kem ic infiltrates ca n be disti ng uis hed from I. Miller .JFAP. Mitehell (;F: Thymus and antigenreactive cells. T ran~plant Rev I ::l 1:.!. Hlfi!l o ne a not her through application of the method 2 Takahashi T. Car:.well EA. Thorhecke C.J Surlace described in this paper Because ot the ease with antigens nl immunncompetent cells I Eifert of which this method ran bt:> performed and the theta and PC . I alluantisera on the a bill! v of spleen ra pid ity with which answers can be obtain Pd from w t rnnsler immunl' responses . .J Exp Med 1:!:.!: II~ I 119(1, l!J70 its use. tt may become a practical aid in the 3. C'laman H:-.J . Chaperon EA: Imm unologic comdiagnosis of cert ain disorders . plementation hetwel'n thymus and marrow Appl ication oft h1s mPthod will allow correlation cells-a model for the two-cell thenr\ of lmbetween tissue set'tton s t udie, of human pat hologic munnwmpetence I r:11v-plant Re\ I :9:l i 1:1. 1969 4. \ 'itettn ES Fliancu G. :\u"en1weig \ , Lhr .J W· Cell proces,.,es and in 'it ro systems through identi!icasurfac(' unmunnglnhuhn IV. Distnhution amonJ! tion ol the effector cell~ in the lesion!-.. During th e thvmol'\'lb. hone marrm• cells. and the1r derived past decade, there have been many studies perJ)(it>ulutions. ,J Exp Mccl 1:16:RI 9:1. 197:! fo rmed which demonstrate that T cells. H celh,, 5. Janossv G. Greav~ MF: l.ymphoc\'te acti\"!ltton. I. Hespnnse ol 'I' and H t\ mphorytes tu phvtomi toand monocyte:. can damage target cells in \'itro . gens. Clin Exp lmmunol9: ~:l 49)1 191! The evidence at present suggests that each oft hese 6. Gcwld HA Pmj!'ress toward u eellular en~:meennl(. d iffe rent cell types may have different ways of .JA:\IA 21 1.1289 1:1o0. Hl70 killin g target cells. For example. whtle T cells from 7. Pem1s B. Porni L. Amante L: Immunoglobulin spots on the surface of rabhtl lvmphocyles. ,J Exp :\led se nsitize d animals can lyse the s ensi tizin g t a rget I:!2: IOU I WlR. 1970 cells, H cel ls from non~ensitized donors can abo 8. Gel) I, Kruger .J. Sp1esel SZ. Sttmulat 1on nt 13-lymmediate speci fi c cv totox ici ty 111 the pre~ence of phon·tes h\• endotoxtn Reaetions ol thymusan ttserum to target cells [28 :101. M o reover. the depriHd mice and kar\ ot\pic analysts nf d1ndmg l'ells 111 mtl'E' heanng T,T, thymus grult....) l mmumacrophage t:. acti,·ated by sensittzed lymphocytes nnl IOlUOAA-1091. l!l";:! through soluble mediators a nd if\ also rendered 9. Craddock GG. Longmtre R, :\1cMillar H Lymcapable of ~pe,·tlic destruction of t a rget cell s in phocytes and the immune resJ)(mse (tirst of two vitro[llj. pa rt~). N I<:ngl ,J Med 28:->::12-t :l:11. 1!17 t We were unabll:' to corroborate the report b} 10 1'\uthun CF, 1\arnovskv :\1 L. Da' id .JR: Alu.•ration» of macrophage functi(.lnh hy medtator-. !rum lym· Silviera. M ende~. and Tolnat [21 I of localization of phocv te,. ,J Exp :\lt>cl 1:1:1:1:t;6 t:l'i6 l911 T cells in lymph node::. by the E test. Since II E'ans R Alexander P Holeol macrnphal!e' m tumor Coombs. Gurner. \\'ilson, Holm. and Lmdgren [:l l I 1mmun1ty. C'oopcrattnn hrtwt'en manophaJ!eS and have s hown that only living cells !!Jrm the nonim lymphoid cells in svn~eneic tumor immunity. l mmunol 2:1:615 626. 1972 mune E roset te with T cells. lymphocyte::; that 12. Hianco C. Patrit·k R. :-.lussen/\H'ig \': A JX>J>ltlatwn ol have undergone lreezinl! and thawing in th e ublymphorytt'S heannl{ a memhrant' ren•ptor for sence of a c ryoprotecti\'e agent would not he antiJ:t' ant ihody-complemPnt compte'e". l ~epa ­ ration and charartenzallon .J Exp Med expected t o bind E in section At present T cells 13:!. 702 720, 1970 must be identified 111 human ussue sectto n by exclusion: mononudear cells which a re not B ce ll s l:l. Huber H. Polle) M.J. Lin~cutt \\"0. Fudenherg HH, MuiiPr·Eherhard H.J: Human monocHes: di;,tincl (IgM EAC pos itive) or monocytes I lgM EAC and receptor slle~ for the th1rd <·ompone1it of t•omplelgG EA positive) are likely to beT cell::;. H owever, ment and for tmmunoglobultn G. Srtenre 1 62:12~1 12~3. l968 14. Abramson .. Gelland E\\', ,Jandl J H. Ho;.en FS: T he • ,J affe E. Berard (', Shp,·arh E. Frank M. Green I. mteratlion betwPen human monontt>" and red Per.,onal communi<·ation cells. Spe<·1firity tor lgG ~ubclasseli and lgG frag-

MONONUCLEAR CELLS I

mentl-..J Exp :\-1 ed l:J2:1:!0i 1:!1;), 1970 l!i. Hu ber II. Dnu)!las sD. Fudenher)! HH : T he l!(C receptor: on tmmunolngtcal marker lor 1he chantcte rtzatwn ut rnononudear cells I mmuuoloj!'y 17:7 :11 . 19lifl 16. Lay \\'11, 1\l ende~ ~F' . Htancu C. :\u!>~en,meil( \ '. Hindin~t ut sheep rl'd blood cells to a lar~te popula tmn nf human J\mphocytes . ;'\ature !Londl :1:10:.1:11 [}:1:1, Hli I 17 .Jondal :1-1. Holm (; , \\'igzell II : Surlaee murke~ on human T and H h mphoc~ te~ . l. A large population ollyrn phontes lorrnml( nonimmunc ro::.elle. \\ilh sheep red blood <·ells . .J Exp :vi ed 1:16:207 21!i, IH7:1 18 Hntl MC : Th<•ta isnnnlll(f'n as a marker nt thymu' deriH·cl lym phocytes m m1cr . l\ature !Lnnd) :!:! t: :I7H :-17!l. 1%9 19. Smith, RW. Te rr~ WD. Hucll U:'\ : An anugen ic marker fnr hu man thymi<· lymphocytes . .) lmrnunol 110:HRI HHi. 1!ln 20. Uukor 1'. Biaru:u (', :>.ussenzwetg \ ': Ti~sue localiza tion of lymphocytes bea nng a membrane receptor lor anul(en -antihnd} -cnmplerne nt torn pl exe~. Pruc :--.. a t I Acad S ci l SA fii:!*ll 997 . 1970 21. Sil\lera l\PA. 1\ltmdes \;f'. Tolnai MEA· Tissue lucahtuunn o l t\\o populations ol h uman lym phnt'\'1p,.. distmgutshed b) membrane receptors . .J l mmunnl 10H· 1J.')(i Jlfi(l , 1972 :?2. Frank :\11\1. Gtu ther ' I The eflect of temperature on the reacti\ itv ut I(Uinea pig complement with II(G and 11(!\.1 haemuhttt antibodies. Immunology 19:91)7 !17 1. l!l70 2:1. Krueger GHF. Herard C\\', PeLelhs HA. Graw Rn , YankeE' HA. Levemhal HG. Hogentine C:\ . Herzig P. Holterman RH . Henderson i'~S: Craft versus hn::.t di~euse · morphologiC' ,·ariat iun a nd diffe rential chngno~1"' 111 l'll(ht case::. ot HL-A matched hone

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marrow trun,.,planlatiun Am .J Pathul 6:J: l79- 19H, 1971 She\'llt' h 1-:l\1. Herhcrrnan R, Frank .\lt\1 , Green 1: Heceptors fo r com1>lement and tmmunuglohuhn on human leukemia t•elb and human lymphohlastoid cell hne~ . .J ('1m lm e.t ;)J :19:3:1- 19:~1'1. Hl7:! Pincus S , Biantn C. ·w.scnt\\eig \ '· ln t rea~ed pro poruun ol cumplement -recep w r I~ mphm·y(e, in the peripheral blood ol patients with chronic lvmphot·~ t l<' leukerniU Hloud -IO::lO:l :110. 19i2 Atsenberg AC, Blot·h K-1 lmm unol(lnhulm" on the surfa<"e ol nt>opfa,ttc lymphucytcs. :\ Eng! .J :\1 ed 287 :!7:1 2i6. 19i~ Brno nll' .)I) , Zucker- Fmnklin D. Weiner MS. Hianro C. 0ju~,.,e n zwcig \ ': Leukemic celb with me mbrane propert ies ufth ymus -derived !T) lym phocy tes in a rase ol SPzary\ s, ndru me: morphologic a nd im· munolol(ic· st udt Ps Chn lmmunnl l mmunnpathol, I 97:1 II n pre~q van Buxel ,JA. l'aul WE. Frank .M :\1 , Green 1: Antihudy dependent lymphoid cell-media ted cytotnxicitv : role ol lvmphnr vles hearin!( a receptor for complemelll .J I mmunol. HJ7:l ()n press! Muller (;, s,chug SE: Specificity ol lvmphocvte rnedtnted ('\'toloXtl'llv mdured bv 111 vitro antl hody t·oated target cell,. Cell lmmunol 1: 1 HI. 19i2 ll enne~ CS. Clayhurl(h .J . ('ule GA. Prenderg;tst RA : H J\·mphncyte medwted t·ywly:;i>-: a complement mdependent phenomenon . lm munol Corn municatums I :H1 10:1. 19';2 Cnomhs RRA. c:urner l:l\\', \\'ibon .-\B. Holm G. Lmdgren B : Ro~ette format inn bet ween human 1\ mpho<'Yll'' a nd ~heep red blood cell~ not 111\'0ivlng rPt·rptors ln t A rth Aller!() Appl lrnmuno l :l!1:6!iH 66:1. IH70