Histol Histopath (1988) 3: 69-80 Histology and Histopathology

lmmunophenotypic characterization of primary and secondary lymphoid follicles Cesar P. de Luaces', Jose I. Pera12, Manuel Garcia Tejeiro2 and Cesar Aguirre2 'Servicio de Anatornia Patologica, Residencia de la S.S. (Carnino de Santiago>), Ponferrada, Leon, and 'Dpto. de Anatornia Patologica, Hospital Universitario de Valladolid, Valladolid, Spain

Summary. Thc need for an immunophenotypical referential framework relative to lymphoid follicle has led us to apply a panel of monoclonal and polyclonal antibodics, by means of a sensitive immunostaining method. Lymphoid follicle is an immunophenotypically complex structure made up of three lymphoid populations (B, being its bulk, and a few T and NK cells). dcndritic reticulum cells (DRCs) and Flemming's macrophagcs. Follicular B population is To 15 +. B1 + , OKB 7 +, HLA-DR + and C3bR +. In secondary follicles therc are differential characteristic reactivities for cach topographic compartment: Mantle zone is positive for OKB 2 and surface IgM (sIgM) and IgD (sIgD): germinal center (GC) clear zone (with centrocytic predominance) for OKT 9, sIgM and weakly for OKB 2: and G C dark zone (with centroblastic predoiniiiance) only for OKT 9. In sections, OKT 10 allows one to see immunoblasts and plasma cells, the latter being with lymphoplasmacytoid cells the only intracytoplasmic immunoglobulin holders. 10% of G C lympliocytcs are T cells, almost exclusively T-helper (Leu 3a +). Another 10% to 15% of lymphoid cells are Leu 7 (HNK- 1) +. In histological sections, DRCs are spccifically marked with R4123 and Flemming's inacrophages with anti-alpha,-antitrypsin and anti- alpha,-antichymotrypsin antibodies, both populations bcing negative to OKM 1 and OKM 5 .

Key words: Immunophenotype - Lymphoid follicle - Iminunohistochemistry

Introduction

Lymphoid follicles are topographic domains of lyniphoid B population (Weissman et al., 1978). There

Offprint requests to: Dr. Cesar Pedro de Luaces de la Herran, S e ~ i c i o de Anatomia Patologica, Residencia de la S.S. &amino de Santiago>,, Ponferrada, (Leon), Spain

are many studies about the immunophenotypic characterization of these structures. either by conventional methods (Curran and Jones, 1977, 1978; Brandtzaeg et al., 1978: Stein et al., 1980; Tsunoda et al., 1980; Curran et al., 1982: Matthews and Basu, 1982; Morris et al., 1983), or by monoclonal antibodies (MoAbs) (Hsu et al.. 1983; Hsu and Jaffe, 1984a. 1984b: Knowles et al., 1984). Nevertheless, there are strong contradictions. specially with regard to thc immuiioglobulins (Igs) (Curran and Jones, 1977, 1978: Tsunoda et al.. 1980; Hsu and Jaffe, 1984b).

In view of thc structural complexity of secondary follicle on light microscopy (Muller-Hermelink and Lennert. 1978). it seems very difficult to assign current reactivities by immunohistochemical techniques. Therefore we have applied a wide panel of monoclonal and polyclonal antibodies on peripheral lymphoid tissues in order to clarify. with the greatest accuracy, the distinctive phenotypes of cellular populations that constitute the secondary follicle.

Materials and methods E

Sections of human tissues obtained from reactive lymph nodes, palatal tonsils, appendices, ileal mucosae and spleens were used.

The aforementioned tissues were embedded in O.C.T. Compound and snap-frozen in 2-methylbutane precooled within liquid nitrogen. Six pm sections wcrc cut and placed on gelatinized slides prior to immunostaining.

In order to visualize several intracellular rotei ins. L - '

comprising various Igs. S-100 protein, alpha,-antichymotrypsin (ACT) and alpha,-antitrypsin (AAT), sections from selfsame paraffin-embedded tissues were employed.

Tables 1 and 2 summarize monoclonal and polvclonal antibodies used in this study, and theirLoptimum concentration for immunostaining procedures.

For staining procedures, the avidin-biotin-peroxidase complex (ABC) technique was used as has been described elsewhere in detail (Hsu and Raine. 1984), using the chromogenic reaction with DABlH,O,

lmmunophenotype of lymphoid follicles

according to Gattcr et al. (1984). A B C reacti\es were obtained trom Vector Laboratorie4.

Results

The negative controls displayed only unspecific stainings 011 sections from fresh-frozen tissues. related \\.it11 endogenous peroxidase activity in eosinophils. l'hcse stainings appeared like a strong intracytoplasn~ic granulate. masking the nucleus.

According to the positivities obtained o n .;ections from fresh-frozen tissues, threc main staining types \\.ere seen: I ) Associated with cellular membrane. 2 ) reticulated or interstitial, and 3) mixed patterns. The thircl t q e . only limited to germinal centers (GC$) , cho\vcd intermediate characteristics between the first a n d second patterns. When polyclonal antibodies and anti-lg b1oAhs were applied to embedded-paraffin section4 onlb. intracytoplasrnic cellular stains became apparent.

The primary lymphoid follicles ancl mantle zone (hlZ) of secondary follicles showed the sarne behaviour \\.hen thcy \\ere marked with different antibodies. Both structures were completel! stained with mcmbrane marker4 to B cells ( O K B 3. O K B 7 . T o 15 and BI ) . IgG. IgM. IgD. and kapp ;~ 2nd lambda light chains (Fig. I ) . They were also positive \vitli T o 5 (C3bR) and H L A - D R .

In the G C of sceondary follicles. the markers for B cells sho~ved a heterogeneous response. A uniform h o m o g e n e o ~ ~ s staining was obtained kvith T o IS and B I (Fis. 3) . whereas OKB 7 and OKB 2 MoAbs conditioned different patterns. The GCs were con~pletely stained \vitli O K B 7 dra~+.ing a mixed pattern. membranous and reticulated. with stronger intensity of staining in the nearest area to the wide part of M Z (Fig. 3 ) . A similar behaviour was shown by T o 5.

While the MZshowed a strong re:rcti\it!. \\.it11 O K B 2. in the G C the positivity obtained ni th this MoAb wa4 weak. The reactivity in G C affected half or two thirds of the G C nearest t o the wide part of M Z (Fis . 4 ) .

In sections of fresh-frozen tissues. IgG was the predominant Ig, being positive throughout the M Z and G C . The kappa predominated over lamhclu light chains. The IgA appeared positive in the nearest area of G C to the wide part of M Z (Fig. 5 ) . This Ig was not re\,ealed in MZ. The IgM was shown positiw in M Z and the nearest area ot' G C to the wide part of M Z (Fig. 6 ) . The IgD r~pl>e:ued only in bIZ, while in G C it was spor a d ' ~c or inexistent (Fig. 7 ) .

Intrac!,toplasmic Igs (cIgs), studied in embedded- paraffin sections, only appeared in plasmablasticl pl;~smac!,tic and very scarce small round cells within G C . The cells clg + were very scarce and they were not always present in all GCs. The cIg prevailing u a s IgG, followcd by sporadic cIgM + ceIls and, in abdominal tissues, some eIgA + cells.

The markers related with D R antigenic con~plcx ( H L A - D R ) stained completely and l~omogeneously all Iymphoid cells in secondary follicles (Fig. 8) .

The presence of transferrin receptor ( O K T 9 + ) was restricted to G C lymphoid cells ancl to :I small amount of

M Z ancl primary follicle lymphocytes (0%) to 15'%1). Also Flemming's macrophages had a higher staining intensit! than lymphoid cells.

With regard to T Iymphoid cell markcr4. r e s ~ ~ l t s \\.ere variable too. Whereas O K T 6 was shown to he \vlioll! negntivc, ivith O K T 1 0 ~ 1 strong staining appearcd in 1070 of G C Iymphoid cells. which had tendenc! to be grouped. Centroblasts and centrocytes mere lightl! 4tninecl \vith O K T 10. The common-peripheral T-cell markcrs ( O K T 3. Leu l . Leu 4, O K T I I and T?) hho\\ed a 10% of positive lymphoid cells. In GC. such cell5 disclosed a strong zonal distribution being rcstrictcd to the nearest area to the wide part of bIZ (Fig. 0 ) . In hlZ. thc ratio between Leu 3a + T-helper cells ( T h ) and OI<T 8 +T-cytotoxiclsuppressor cell4 (Ts) \\.as hrpt to 211. Nevertheless. in G C that ratio raised up to ThIT5 = I'll (Fig. 10).

In G C a 10% to 15% of Leu 7 ( H N K - 1 ) + Iymplioitl cells with homogeneous distribution \\.as euclu4i\.cl! rcvealed (Fig. 11).

With R4123 and FHC17 MoAbs a follicular net\\ork \vas drawn without Iymphocytic rnemhranc pattern. displaying a stronger staining for first of them (Fig. 13). The O K b l I and OKM 5 MoAbs, in sections of frcsh- frozen tissucs. and anti-S-100 protein, in embedded- p;~raffin sections. did not show reactivity in I!mphoid follicles. Flemming's ~nacrophages had a cytoplasmic positivity for A C T and A A T in the latter sections.

J5 showed negative, or very weakly positive. staining in GC$.

Fig. 1. IgD + primary follicles in a lymph node. Countersta~ning w ~ t h haematoxylin, t 40

Fig.2.Bl + secondary tonsil follicles. Countersta~n~ng wlth haernatoxylin. X 100

Fig. 3. OKB 7 + secondary follicles in a lymph node. A light zonal tendency for the staining in central follicle can be observed. Counterstain~ng with haernatoxylin. X 100

Fig. 4. Secondary lymph node follicles marked with OKB 2. Counterstaining with haematoxylin. X 100

Fig. 5. Heal mucosa. IgA positivity is shown in enteric epithel~um and GC, in its nearest portion to the lumen. Counterstaining with haernatoxylin. X 100

Fig. 6. Secondary tonsil follicle with good development and typical positivity for IgM. Counterstaining with haernatoxylin. * 100

Fig. 7. Two secondary lymph node follicles wlth IgD i- MZ. Counterstainlng with haematoxylin. X 100

Fig. 8. Secondary lymph node follicles marked with anti-HLA-DR. In T-zone are positive interdigitating reticulum cells too. Countersta~n~ng with haematoxylin. X 100

Fig. 9. Lymph node stained with Leu 3a. In center, asecondaryfollicle with few Leu 3a + cells, mainly in right half of GC. On the right of figure IS the T-zone showing intense positivity. Counterstaln~ng with haernatoxylin. r 100

Fig. 10. Lymph node stained with OKT 8. The GC IS completely unprovided of Ts-cells. Counterstalning with haematoxylin. X 100

Fig. 11. Leu 7 (HNK-l) + cells in a foll~cular lymph node GC Counterstaining with haematoxylin. X 100

Fig. 12. Secondary lymph node follicles positive for R4123 showing a network of DRCs. Counterstaining with haematoxylln. X 100

lmmunophenotype of lymphoid follicles

Table l. Specificity, Optimum Work Concentration and Source of Monoclonal Antibodies.

Antibody Specificity Work Concentration

OKT 6 OKT 10

OKT 3 OKT l l T2 Leu l Leu 4 Leu 3a OKT 8

OKB 2

OKB 7 To15

Leu 7 (HNK-1)

OKM 1 OKM 5 FHC17

OKT 9 J5

Thymocytes Stem Cells. Thymocytes. Prothymocytes, Null Cells, Monocytes (+/-), Activated Antigen TCells E Receptor T Cells T Cells T Cells Helper/inducerT-subset Suppressor/cytotoxic T-subset slg + BCells, Squamous Epithelium, Granulocytes slg + B Cells B Cells B Cells Delta Heavy Chain Mu Heavy Chain Fc Fraction of IgG Alpha Heavy Chain Kappa Light Chain Lambda Light Chain Large Granular Lymphocytes, NWK Cells, Some Leu 2 + Cells Monocytes, Granulocytes Monocytes, Platelets Monocytes, Macropahges. Langerhans Cells Dendr~tic Reticulum Cells C3bR Beta Chain of HLA-DR Molecules Transferrin Receptor Common Acute Lymphoblastic Leukaemia Antigen

Sources

Ortho Diagnostic Ortho Diagnostic

Ortho Diagnostic Ortho Diagnostic

Dakopatts Becton-Dickinson Becton-Dickinson Becton-Dickinson Ortho Diagnostic

Ortho Diagnostic

Ortho Diagnostic Dakopatts

Coulter Clone Dakopatts Dakopatts Sera-Lab

Miles-Yeda Sera-Lab Dakopatts

Becton-Dickinson

Ortho Diagnostic Ortho Diagnostic

Sera-Lab

Dakopatts Dakopatts Dakooatts

Ortho Diagnostic Coulter Clone

Table 2. Specificity, Optimum Work Concentration and Source of Polyclonal Antibodies

Antibody Specificity Work Concentration

Sources

Anti-alpha,-antitrypsin Monocytes and and Histiocytes 1 /500 Dakopatts (Kerdel et al., 1982)

Anti-alpha,-antichymotrypsin Monocytesand Histiocytes 11500 Dakopatts (Kerdel et al., 1982)

Anti-S-l 00 protein Langerhans Cells, 1 /400 Dakopatts lnterdigitating Reticulum Cells (Kahn et al., 1983)

lmmunophenotype of lymphoid follicles

Discussion

The secondary lymphoid follicles n1.c phcnotypic~rlly assymetrical structures made lip of n GC' a n d a sur~.ouncling Iynphoid ring namecl m:rntlc /one. The GC' pre5cnts :I lighter stainccl xrea (clear /one). close to the wiclc part of MZ with ccntrocytic predominance. ant1 o the opposite side a clark st~rincd area with ccnt~.ohlastic 1)rccdominance (Muller-Hcrmelink and Ixnncr t . 1078). Our findings reveal that surface markers tbr B cclls and Igs beh;r\,c hcterogencousl~~. ancl that a follicular :rssy~lwtry is mantained when imm~~nol~istocliemic:~l t cchn iq~~es are applied. The complcxit! of this structure incrcasc \vith the presence of other T and NK Iymphoicl cells and reticular cclls.

Whereas with T o 15. R I . and anti-DK system antibodies homogeneous follicular staining is olltuined. the results with To 5, OKB 7 and OKB 2 are different.

Apl'lying T o 5 (anti-C'3hR) and OKB 7 ILloAba. the M Z I!,mphocytes are uniformly stained. hut in the GC a n ass!.lnctric ancl parti:rllq' reticulated staining is seen. Other investigators have rccognirccl C'.; frirction rcceptors in GC' lyrnphoid cells h!, mcans of rosctting techniclucs (Stein et al . . 1080). and in clendritic I-cticular cclls (DKCs) by irnrnunoseru (Rqnc.4 et :(I.. 1985). Ho\vc\.er. with SLICII ser;r Tsunoda et al. ( 1080) I'ound the aforcmentioncd reccptor\ only in 500;, 01' GC' I>,mplioid cclls employing n more specific mcthocl than tliat of resetting. The double presence of po\iti\,it! for Iymplioid cell\ and DRCs should explain the ~ i i i ~ c c l pattern of GC' staining.

I t is \veil knukvn that OKR 7 and O K B 2 h1oAhs are po\itivc in a l~nost the totalit! of sIg + I~mphoicl cells oht:rinccl from blood ancl cellular \u\l)ensions f r o ~ n Iymphoicl tissues (Mittler et al.. IOS.7 I . In seconctar) follicle. with O K B 7. \ve lia\.c oht:rined a stainin? himilar to tliat of T o 5. Therefore. this fact lead 11s to consider OKB 7 a marker for Iymphocytcs oncl IlKC's. KnowIcs et al. ( 1084) corroborate the rcacti\itv for OKR 7 in GC Iymphoid cells. but do not dcscl-il,c this rc:rction in DRC's. However. some of our ca\e\ cliagno\cd as ccntrohlastic-centl-ocytic follicula~. I>rnpho~n:r. \\.hose malignant cclls were OKB 7 unreac t i~c . clearly sho\vcd OKB 7 + DRCs ( D e Luaccs et :(I.. 1987). Tlic , "re:rter clcnsity of DRCs in G C clear zone should explain tlie stronger staining intensit!, obtained in this zone with tliis Mo.Ah.

In the preccnt study, O K B 2 h:rs sholvn po.;iti\,ity in MZ. ancl a li9ht staining in tlie clc:rr zonc of the GC'. Thcrefore. this MnAh spares thc portion of c c ~ i t r ~ l ~ l a s t i c prcdorninnnce where the GC' proliferative activity is >,icltlcd (Stein et al.. 1085). Thi\ light staining\vith ~ o n a l ~~~~~~~~n obtained in thc space of centrocytic predominance does not confirm tlie assertion th~rt O K B 2 is a true pan-B m;rrker(Kno\vlcs et al.. 1084: Kno\vle\. 1985). Moreover. i t is not estahlishcd that thi\ markcl. must stain DRCs.

With regard to Igs. 0111- r e s ~ ~ l t s II:I\.C been \.cry tliffcscnt tlependinr! on if the tis\ucs were fresh-frozen or paraffin-embedded. Although there arc sornc palxrh

\vliich agree that predominant Igs in the GC' are oi' IgC; tylw (Brantltzacg et al.. 1078: Curran and Joncs. 1977. 1978). tlie interpretation of thcir cellul:rr specificities is contro\ersial. There are reports recording G C Igs being extracellular (Curr~un and Jones. 1977: Hsu et al . . 198.7: Hsu and Jaffe. 1984b). whe~.e:rs in other ones a significant amount of slg t lymphoid cells is rccorclcd (Curran a n d Jones. 1978: Tsunod:~ et al.. 1080). Mo5t Igs. mainly I8G. probabl! make up part of electron-clen\c rnatcr~nl placed bctwecn the cytoplarnic processes of the DRCs (M~~l le r -Hcrn~e l ink ancl L.ennert. 1078).

The rcwarchers agree that the M Z I!pmpl~ocytes arc \IgD + and sIgM +. Ho\vevcr. the GC cxprcsscs sIgh/l mainl!, in the clear zonc and IgD is absent in that s t r ~ ~ c t ~ ~ r e . We conclucle that IgM is predon~inantl> cxpressccl o n the centrocytic cell surface. unlike other reports stating that slg is found only in vcr!. >cant!, G C cclls (Hsu et al.. 1083). pal-ticularly centroblasts ( H ~ L I and Jaffc. 1c)S-lb). Ncverthcless. o ~ ~ r opinion is in agreement ni t11 some previous experimental works. So. it ha5 heen shown. in cnuclcated lymphoid follicles from lx~latal tonsil\. that half of G C large Iymphocytea are slg- bearer (Tsunoda et al., 1980). Furthcrmorc. \mall Iymphoc~~tcs s i m ~ ~ l t a n e o ~ ~ s l y hearing 41fD :IIKI slghl. ancl large Iymphoid cells hearing only slgM arc seen by means of separation studies from r n ~ ~ r i n c splccn cells (Gootlman et al.. 1V75). l'hcreforc it does not seem strange that a wide amount of centrocytes are slg-hearers. nhcrcas centroblasts are sIg-negative. This idea is concosclant ~vith the findings obtained in studies about Ig expression in 11~1nlan lymplioid cell lines. D ~ ~ r i n g their de~.clopment. these cclls attain an irnm~~noglobulinic phenotype corrcspo~~ding to ;I mature B cell (slghl + ancl \IgD +) . Thereafter they complctcly loose their ininiunoglobulinic expression. regaining it suhsccluentI> like n pre-13 cell (sIgM +) (Gugliemi and Preud'Homc. l~As1 1.

In relirtion to clgs. our result\ are in concordance with those of other in\,cstigators with rcgarcl to the scanty 1"-cscnce (01' c1g + cells in GC. hcing clearl>- predominant clgG (Tsunoda et al.. 1980; Matthens :und B ~ S L I . 1082: Brundtzaeg ct al.. 108.7: Morris et ; I ] . . 108.;: HSLI and Jaffe. 1984b). These cells mainly correspond to tlie Intc stages of plasma cell differentiation. \\,hereas normal irnniunoblasts lack aforementioned cl: (klorri\ et al.. 1083). Howe\.cr. HSLI ancl Jaffe (IOS4h) report some clg + centrocytes. \hlc belie\.e that 5cnrc.e cIg + small Iymphoid cells arc Iymphoplasmacytoitl ones. The latter. ~111likc ccntl-oqtes. 1i;rve C O ~ ~ O L I S r o ~ ~ g l i cnt1opl:rsmic rcticulurn (Muller-Hcl-ntclinli and Lennert. 1978).

With regard to results obtainecl by means of nnti-T cell MoAhs. trio dil'l'crent e \ j c ~ ~ t s O C C L I ~ : O K T 10 positi\,it! for B cclls in GC. :rntl presence of an a c c o m p : i i T ly~nplioid population ~vitliing G C .

The O K T l ( l antibod! has been rclmrtecl like rcacti\.e for B cell population. when this i \ mirturing to \ \a~-ds plasma cell and los in~othersurface B markers. O K R and B1 (blittler et al.. 1983). Previously. O K T 10 + cells has

lmmunophenotype of lymphoid follicles

hccn tlc\cl.ihed i n \\.hole GC'. \\;it11 :I greatcr. positi\.it!, in clcar /c)~ lc ( l 1\11 ;111c1 J:~i'fe. L984;1). N e \ ~ e r t l ~ e I c ~ s . 0111.

result\ r lcnote that OKT I 0 is \veakl!; rc:~cti\.e \\it11 ccntroc!te\ ant1 ccntrohl:~sts. \vhcteas i t i x str-ongl! po \ i t i \ c for. imm~~noh l ;~s t \ / p l asma cells.

In ~rc lat ion Lo presence o f T I>~~npllac!.tcs i n GC'. there i\ :I \tl 'ong preclomin:~cc o f thc\e in c1eu1- zone. mainl! I ~ c i n g :I T - I ~ c l p c ~ ~ ~ ~ o p u I a t i o n (Si et al. . 1983). I ' l i is f ind ing conf i rn i \ the assymctric nature of sccondar! (c)lliclcs. 'l'licis I > ~ C \ ~ I I C C corrohos:~tcs the importancc o f T-B c o o l w s ~ i o n ( J l c M i l l ; ~ n et al. . 1981 ).

.l'lic ol,\el.\:ttion of L c u 7 ( H N K - l ) + cel l \ i n GC' \liggesL\ [hat {hi\ populat ion can hc rcl:~tccl \\,it11 the contl-01 of GC' c lc \e lo l~ment . This same e\,cnt ha\ hecn \ i ~ g g c \ ~ c d h\ other in\ 'cst igators (S\verdlo\\ ~ ~ n d hlurra!.. lOS4).

~ Z m o n g ~ ' c t i c ~ ~ l a r cells :~ssoci:~tcd t o I!vnphoid foll icles. l l I i ( ' \ ancl F lcmming's mactopliages. the fo rmer is \pcc i f l icall! m;irkccl \\,ill1 K4133 MoAh. Since DRC's arc p o i i t i \ c f o r FlIC'I 7 too. t l i cn \VC s h a ~ l l d allege for t hcm :I monon~~c lca r /p l~ i~goc !~~ ic s!stcm or ig in. such as otl icl . \\ I-iter\ l i ; ~ \ c suggcstcd (Gc rdcs et al.. 1083). Hawc\ .c r . the! do no t \ho\\.n rcacti\:it! f o r OKb1 1 an(l OKhl 5 (h lo .Ahs LO soluhlc antigen ~ ~ c s c t i t i n g monoc!,tic/ rnac~.ol'hagic\ystcm cells (Sl icn et al.. IC)S3)). T l i c lack 0 1 l.c.;~c~i\ it!. I'os OK\l I ancl OKbI 5 s~~ggcs t s that LIRC's m:\! h;~\ c ;I mc\cncIi!.~ii;~I 01-igi11. ~~~ i r c : I ; ~ tec I t o ~ ~ i o ~ i o c ! tic/ m ; ~ ~ ~ ~ - o p l i a g i c \ ! \ ~ c m . \ \~I i ich is in ;~gl.cerncnt \ \ . i ~ l i p r c \ i o l i \ LII~~;I~~I.LIC~LII-;II s t~ ld ies I'rom de\~c lop ing l ymph noclc.\ (C ; ro \c~~r t l i . 19511: S a k ~ i m a et al.. 1081 : h Ia rkg~.a f c t al. . Ic)S2).

I n rc l ;~ t ion t o I. ' lcmming's mac~.ophagc. this is a nionoc!tic/m;~csopliagic s!,stcm cell \\-it11 i t r o n g ph;~goc!.tic cfl'icicnc!. ( h l u l l c r -Hc r l nc l i nk nnrl Lcnnc r t . 1075: Lashcr. lOS.7). 7'his cell lacks antigen prcscnt ing ~,a l>;~ci t \ \ \h ieh is c lc~nonstratcd by nn~.cacti\,it!. 1.01- OKhI I ancl O K L I i.

T o \unimar i /c . this stucl\ has sllo\vn that \cconclar! ~ o l l i ~ l c \ ;II-c \~~LIC~LII.CS \\.it11 c o ~ n p l c x a n d ass!-mettic i m m ~ ~ n o ~ ' h c ~ ~ o t ! , p c . ant1 i t has propo~' t ionccl a rct'crenti:11 t r a ~ i i c \ \ o r h to in\.estigatc t l ic i1nrnunol7llcnot!.l7ic:1l niolli l ' ic:~rion\ of n o d ~ l l a r l!,mphomas. b lo rco \ ,c r . some l ' i~~c l ing \ . \ \h ieh \ \ c ~ - c not descrihcd o r cmpli:~si;.ed ~ ~ - c \ i o ~ ~ s l ! . . \ \ere sho\\n. I'oI- instance: s l gb l + cc,lls (~> rec lom i~ i i ~n t l ! ccntroc! tcs) are niainl! placecl in clc.211- zonc o f (;C'. I7ut no t i n dark zonc: OKB 2 is no t :I pan-I3 cel l r n a ~ k c r \incc t lark zone o f GC is ~ ln l .cact i \ ,c : ancl O K H 7 J,Io,Al, is rc;~ct i \ .c \\.it11 DRC's too .

References

Brandtzaeg P,. Surjan L.Jr. and Berdal P. (1978). lmmunoglobul~n systems of human tonsils. I. Control subjects of various age: Quant~ficatlon of Ig-producing cells, tonsilar morphometry and serum lg concentrattons. Clin. Exp. Immunol. 31, 367- 381.

Curran R.C. Gregory J. and Jones E.L. (1982). The distribution of ~mmunoglobulin and other plasma protelns in human reactive lymph nodes. J. Pathol. 136.307-332.

Curran R.C. and Jones E.L. (1977). Immunoglobul~n containing

cells in human tonsils as demonstrated by ~mmunoh~stochemistry. Clin. Exp. Immunol. 28, 103-1 15.

Currran R.C. and Jones E.L. (1978). The lymphold follicles of the human palatlne tonsil. Clln Exp. Immunol. 31, 251-259.

De Luaces C.P.. Peral J.I. and Mateos J.J. (1987). Linfoma follcular con lmagen de -clelo estrellado>>: a proposito de un caso. Patologia (submitted).

Gatter K.C.. Falini B. and Mason D.Y. (1984). The use of monoclonal antlbodles in histopatholog~cal diagnosis. In: Recent advances in histopathology, no 12. Anthony P.P and MacSeeen RNM (eds.) Church~ll Llvingstone. New York. pp. 35-67.

Gardes J.. Stain H.. Mason D.Y. and Ziegler A. (1983). Human dendrlttc reticulum cells of lymphoid follicles: Their antigenic profil and their identification as multinucleated giant cells. Virchows Arch. (Cell Pathol.) 42, 161-172,

Goodman S.A.. Vitetta E.S., Melcher U. and Uhr J.W. (1975). Cell surface immunoglobulin. XIII. Distribution of IgM and IgD like molecules on small and large cells of mouse spleen. J. Immunol. 1 14.1646-1 648.

Groscurth P. (1980). Nicht-lymphatische zellen in der lymphknotenrinde der maus. II. Postnatale antwicklung der interdlgitierenden zellen und der denditischen retikulumzellen. Path. Res. Pract. 169,235-254.

Gugliemi P. and Preud'Homes J.L. (1981). Irnmunoglobulin expresion ~n human lymphoblastoid cell lines with early B cell features. Scand. J. Immunol. 13,303-31 1.

Hsu S.M., Cossman J. and Jaffe E.S. (1983). Lymphocytesubsets in normal lymphoid tissues. Am. J. Clin. Pathol. 80, 21-30.

Hsu S.M. and Jaffe E.S. (1984). Phenotypic expression of B- lymphocytes. 1. ldentlfication with monoclonal antibodies in normal lymphoid tissues. Am. J. Pathol. 114,387-395.

Hsu S.M. and Jaffe E.S. (1984). Phenotypic expression of B- lymphocytes. 2. lmmunoglobulin expression of germinal center cells. Am. J. Pathol. 114,396-402.

Hsu S.M. and Raine L. (1 984). The use of avidin-biotin-peroxidase complex (ABC) in diagnostic and research pathology. In: Advances in immunohistochemistry. DeLellis R.A. (ed.). Masson Publ~shing U.S.A. New York. pp 31-42.

Kahn H.J., Marks A.. Thom H. and Baunial R. (1983). Rols of antlbody to S- l00 protein in diagnostic pathology. Am. J. Clin. Pathol. 79,341 -347.

Kerdel F.A., Morgan E.W.. Holden C.A. and MacDonald D.M. (1982). Demonstration of alpha,-antitrypsin and alpha,- antichymotrypsin in cc~taneous histiocytic infiltrates and a comparison with intracellular lysozyme. J. Am. Acad. Dermatoi. 7. 177-1 82.

Knowles D.M. 11. (1985). Lyrnphoid cell markers. Their dlstribution and usefulness in the immunophenotypic analysis of lymphoid neoplasms. Am. J. Surg. Pathol. 9 (suppl.), 85-1 08.

Knowles D.M. II, Tolidjlan B., Marboe C.C., Halper J.P., Azzo W. and Wang C.Y. (1 984). Distribution of antigens defined by OKB monoclonal atlbodies on benign and malignant lymphoid cells and on nonlymphoid tissues. Blood 63, 886-896.

Lasser A. (1 983). The mononuclear phagocytic system: A review. Hum. Pathol. 14, 108-1 19.

Markgraf R., Von Gaudecker B. and Muller-Hermelink H.K. (l 982). The development of the human lymphyd node. Cell Tissue Res. 225, 387-413.

Matthews J.B. and Basu M.K. (1982). Oral tonsils: An ~mmunoperoxidase study. Int. Arch. ~ l l e rgy Appl. Immunol.

lmmunophenotype of lymphoid follicles

69.21 -25. McMillan E.M., Wasik R. and Everett M.A. (1981). Identification of

T-lymphocytes and T-subsets in human tonsil using monoclonal antibodies and the immunoperoxidase technic. Am. J. Clin. Pathol. 76, 737-744.

Mittler R.S., Talle M.A., Carpenter K., Rao P.E. and Goldstein G. (1983). Generation and characterization of monoclonal antibodies reactive with human B lymphocytes. J. Immunol. 131, 1754-1 761.

Morr~s H.B., Mason D.Y. Stein H. and Lennert K. (1983). An immunohistochemical study of reactive lymphoid tissue. Histopathology 7,809-823.

Muller-Hermelink H.J. and Lennert K. (1978). The cytologic, histologic, and functional bases for a modern classification of. lymphomas. In: Malignant lymphomas other than Hodgkin's disease. Histology, cytology, ultrastructure, immunology. Lennert K. (ed.). Springer-Verlag. Berlin. pp. 1-71.

Reynes M,, Aubert J.P., Cohen J.H.M.. Audouin J., Tricottet V., Diebold J. and Kazatchkine M.D. (1985). Human follicular dendritic cells express CR1, CR2 and CR3 complement receptors antigens. J. Immunol. 135,2687-2694.

Sakuma H., Asano S. and Kojima M. (1981). An ultrastructural study of the primary follicle in the lymph node. Acta Pathol. JPN. 31,473-493.

Shen H.H., Talle M.A., Goldstein G. and Chess L. (1983).

Functional subsets of human monocytes defined by monoclonal atibodies: A distinct subset of monocytes contains the cells capable of inducing the autologous mixed lymphocyte culture. J. Immunol. 130,698-705.

Si L., Roscoe G. and Whiteside T.L. (1983). Selective distribution and quantifitation of T-lymphocyte subsets ~n germinal centers of human tonsils. Definition by use of monoclonal antibodies. Arch. Pathol. Lab. Med. 107, 228-231.

Stein H., Gatter K., Asbhar H. and Mason D.Y. (1985). Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies. Lab. Invest. 52,676-683.

Stein H.. Grosskopf E. and Lennert K. (1980). Demonstration of C3 receptors on frozen sections of normal lymphoid tissue and malignant lymphomas using various EAC complexes. Blut 41, 101-118.

Swerdlow S.H. and Murray L.J. (1 984). Natural killer (Leu 7+) cells in reactive lymphoid tissues and malignant lymphomas. Am. J. Clin. Pathol. 81,459-463.

Tsunoda R., Yaginuma Y. and Kojima M. (1980). lmmunocytological studies on the constituent cells of the secondary nodules in human tonsils. Acta. Pathol. JPN 30. 33-57.

Accepted September 8, 1987