Immunology 1986 59 411-417

The primary mediastinal clear cell lymphoma of B-cell typehas variable defects in MHC antigen expression

P. MOLLER,* B. LAMMLER,* B. HERRMANN,* H. F. OTTO,* G. MOLDENHAUERt & F. MOMBURGt*Institute of Pathology, University of Heidelberg and tInstitute for Immunology and Genetics, German Cancer Research Centre,

Heidelberg, West Germany

Acceptedfor publication 26 June 1986

SUMMARY

Eight cases of the recently reported 'primary mediastinal clear cell lymphoma of B-cell type' (Molleret al., 1986) were examined immunohistologically for the expression of cytoplasmic and/or surfaceantigens ofMHC class I and II with mAbs directed against framework determinants of HLA-A,B,C(W6/32; B9.12.1), HLA-DP,DR,DQ (2 06), -DQ (Leu 10; Tii22), -DR (Tu34) gene products, andwith mAbs specific for 2-microglobulin (BBM-I) and the HLA-D associated invariant chain (Vic-Y1). Besides the reported Ig-deficiency, the neoplastic B-cells of 7/8 tumours have variable defects inMHC antigen expression. Three lack both class I and class II antigens, one tumour lacks class Iantigens but expresses HLA-DQ and - DR on the majority of neoplastic cells, three others containvarying proportions ofMHC-antigen deficient tumour cells. The expression of Ii is closely correlatedwith HLA-D(R) expression and its antigenic sites are strictly located in the cytoplasm. Against thebackground of current knowledge, the variable and occasionally severe defects in MHC antigenexpression within the herein presented series of B-cell lymphomas suggest that this unusual featuremight be another characteristic of a novel lymphoma type.

INTRODUCTION

Besides immunoglobulin and other lineage-restricted, differen-tiation-associated antigens (reviewed by Nadler, 1985), normalperipheral B cells abundantly express MHC class I and IImolecules at their surface. Class I antigens serve as restrictionelements for cell-mediated immune response (Zinkernagel &Doherty, 1979; Sanderson & Beverly, 1983; Evans & Engleman,1985). On B-lymphocytes class II antigens take a crucial part inantigen presentation, activation, and maturation (Palacios,Martinez-Maza & Guy,'1983; Bonagura et al., 1985; Corely etal., 1985; Cambier et al., 1985). In neoplastic B cells, however,deficient expression of Ig might occur more frequently thaninitially assumed, since most of the so-called 'null' cell (i.e. Ig-/E-) lymphomas seem to be of B-cell origin (Horning et al., 1984;Gregg et al., 1984; Knowles et al., 1985). In contrast, partialdefects in MHC antigen expression seem to be a rare phenome-non known to exist in some B-cell lines (Rosa et al., 1983; Long,Mach & Accolla, 1984; Accolla, Carra & Guardiola, 1985) and

Abbreviations: AEC, 3-amino-9-ethylcarbazole; DMF, N'N-dimethylformamide; E, sheep erythrocyte receptor; H, Harris' haema-toxylin; 1g, immunoglobulin; Ii, HLA-D associated invariant chain;mAb(s), monoclonal antibody(ies); MHC, major histocompatibilitycomplex.

Correspondence: Dr Peter M6ller, Pathologisches Institut derUniversitiit, Im Neuenheimer Feld 220, D-6900 Heidelberg, FRG.

in a small proportion of peripheral B-cell lymphomas (reviewedby Guy, Krajewski & Dewar, 1986a).

We recently described a group of unusual mediastinaltumours that occurred in young adults, predominantly females(M6ller et al., 1986). Their common feature was 'clear cell'morphology, B-cell nature as demonstrated by the presence ofthe B- I antigen (Stashenko et al., 1980) and complete Igdeficiency. Concerning the clinical data, there are strikingsimilarities of our series with the 'diffuse large cell andundifferentiated non-Burkitt's lymphomas with prominentmediastinal involvement and poor prognosis' that was pre-viously described by Trump & Mann (1982), however, notascribed to the T- or B-lineage. We suggested, that the primarymediastinal clear cell lymphoma of B-cell type is a novel B-lymphoma variant.

In the present article we report that seven out of eight of theB-lymphomas studied have variably severe defects in MHCantigen expression.

MATERIALS AND METHODS

PatientsDuring the last 2 years, eight unusual primary mediastinaltumours were collected that occurred in young adults (19-43years, mean: 29-4), predominantly female (6/8) (see Table 2).The tumours consist of pleomorphic cells with clear cytoplasm

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w-~~~~~** 'I .

Figure 1. Overview on the histological structure of the mediastinal clear cell lymphoma of B-cell type: mostly diffuse growth pattern;light appearance of the tumour caused by the leptochrome and mostly abundant cytoplasm of the pleomorphic neoplastic B cells;numerous mitoses and single cell degenerations; typical ill-defined 'piece meal' necroses (arrows). Depicted is case no. 3 the cells ofwhich completely lack MHC-antigens (paraffin section; haematoxylin/eosin; x 131).

varying in size and nuclear morphology (Fig. 1). It could beshown that they are B-cell neoplasms with a characteristicimmuno-phenotype: leucocyte common antigen+, common

acute lymphoblastic leukaemia antigen-, B1-antigen+, Ig-(surface and cytoplasmic). All patients except one (who has beenin complete remission for the past 15 months after CHOPregimen) responded badly to aggressive anti-neoplastic therapy.None ever developed leukaemia, but abdominal spread ispresently suspected in two patients; four died I1, 13, 18, and 22months after diagnosis. Further details on clinics, histomorpho-logy, cytokinetics, and immunohistology are published else-where (Moller et al., 1986).

Tumour tissuesFresh tissue was transferred immediately after surgical removalto our laboratory. Representative samples were quick frozen inliquid nitrogen. Frozen sections (4-6 ,um) were air-dried over-

night, subsequently fixed in acetone for 10 min at room

temperature and immunostained immediately or stored at - 20°for a short period.

ReagentsThe mAbs against MHC antigens used in this study are listed inTable 1. They are kind gifts from the producing laboratories (seethe References in Table 1) except for Leu 10 that was purchasedfrom Becton Dickinson (Mountain View, CA). Monoclonalantibody binding was detected with a polyclonal biotinylatedsheep antibody to mouse immunoglobulins and a streptavidine-biotinylated peroxidase complex (Amersham Buchler,Braunschweig, FRG). Pooled human immunoglobulin ('y-Venin') was supplied by Behring (Frankfurt, FRG). 3-amino-9-ethylcarbazole (AEC) and N'N-dimethylformamide (DMF)were obtained from Sigma (St Louis, MO).

Staining procedureMabs in culture supernatants were used undiluted; ascitespreparations were utilized as 1:200 dilutions; Leu 10 was diluted1: 50, the anti-mouse Ig antibody 1: 50, and the streptavidine-peroxidase complex 1: 100. All dilutions and washing steps werecarried out in phosphate buffered saline (0- 1 M; pH 7-4), thesecondary antibody solution contained 5% pooled human Ig toinhibit cross-reactions with human surface Ig. Incubation timeswere 1 hr at room temperature for the primary antibody and 30min each for the second and third step reagent. Using AEC asthe chromogen (0-4 mg/ml in 0 1 M acetate buffer, pH 5 0, with5% DMF and 0-01% H202 for 10 min), the peroxidase reactionresulted in an intense red precipitate. The sections were rinsed intapwater, counterstained with Harris' haematoxylin andmounted with glycerol gelatin. Intrinsic positive controls for theimmunoreactivity in each section were interstitial dendritic cells,endothelial cells and macrophages that indicated by theirstaining the reliability of the reaction and thus excluded false

Table 1. List of antigens detected and of primary antibodies usedin this study

Antigen Clone Reference

HLA-A,B,C W6/32 Barnstable et al., 1978B9.12.1 Malissen et al., 1982

f2-microglobulin BBM.1 Brodsky, Bodmer& Parham, 1979HLA-DR,DP,DQ 2-06 Charron & McDevitt, 1980HLA-DQ T022 Ziegler et al., 1982HLA-DQwl, w3 Leul0 Brodsky, 1984HLA-DR Tu34 Pawelec et al., 1982Invariant chain VIC-Y1 Quaranta et al., 1984

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Table 2. MHC antigen expression of the tumour cells in eight cases of primary mediastinal clear cell lymphoma of B-cell type

1* BM 2 FM 3 WI 4 LI 5 GS 6 SS 7 KM 8 SGfem. l9y fem. 21y fem. 31y fem. 35y fem. 35y fem. 43y male 22y male 29y

Clone t: 22mo t: lOmo t: 11 Mo t: 18 mo t: 13mo t: 18 mo c.r.

HLA-A,B,C W6/32 - + - +/- _21 > +3.1 - +XB9.12.1 +/- + - + _ + - + 4.1

12-M BBM.1 +/- + - + > _ - > + - +/-

HLA-DR,DP,DQ 2 06 +/- + - + >>-+> 2.2 _> +3.2 - +HLA-DQ T022 +/- + - + >- + >- - -> +HLA-DQwl, w3 LeulO +/- + - + >- -> + - -> +HLA-DR T034 +/- + - +>- + > > + +4.2Ii Vic-Yl+I- + - + >- +2.3 > + - +

* Case number, initials, sex, age at diagnosis, t death occurring x months thereafter (c.r. =complete remission). Symbols used: +, expression ofantigen on the majority of cells; + /-, positive and negative tumour cells in equal parts; + > -, more positive than negative cells; - > +, very fewscattered positive cells. 1- Situation depicted in Figs 1-4.

t This case contains scattered positive large cells, probably histiocytes.

negative results. Negative controls were performed by omittingthe primary antibody. No staining was observed, except for thereaction ofintermingled granulocytes whose endogenous perox-idase was not destroyed.

RESULTS

The results of the immunoreactions concerning the tumour cellpopulations are listed in Table 2.

Class I antigens

The mediastinal B-cell lymphomas presented here showed aremarkable heterogeneity in class I antigen expression. Only onecase expressed HLA-A,B,C and f2-microglobulin in accordancewith the orthologous situation. Three cases lacked class Iantigens completely (Fig. 2.1). A further case (no. 6) containedonly a small population of scattered class I antigen-positivelarge cells (Fig. 3.1). These were probably intermingled histio-cytes, although it could not be excluded that they belonged tothe tumour cell population. The three remaining cases exhibiteda lack ofclass I antigen expression in a variable but considerableproportion of the tumour cells (Fig. 4.1). There was a markeddifference in binding of the mAbs W6/32 and B9. 19 in case no. 1where B9. 19 bound to about half of the neoplastic lymphocytesbut W6/32 failed to react. In case nos 4 and 6, BBM-l as areagent for #2-microglobulin recognized more tumour cells thanthe HLA-A,B,C framework antibodies W6/32 and B9.19.

Class II antigens

In this lymphoma group we observed striking variations in classII antigen expression. There was expression ofclass II moleculeson all the neoplastic cells in only one case (no. 2). In two casesthe neoplastic B-cells were completely devoid of class IIantigens. Case no. 6 contained scattered HLA-DR positive largecells (Fig. 3.2) that did not react with T022 and LeulO. Theywere probably not part of the neoplastic cell pool but rather

histiocytic infiltrates of monocytic origin. This view would alsoaccount for the non-coordinate expression of - DR and - DQ,since it was recently shown that the myelomonocytic lineagenormally exhibits considerable differences in expression of thesemolecules (Alonso et al., 1985; Edwards et al., 1985; Aglietta etal., 1986; Sparrow & Williams, 1986). Case no. 1 contained classII antigen-positive and antigen-negative tumour cells in equalparts and in case no. 4 class II positive lymphoma cells were themajority. In the remaining cases the situation was morecomplex: the neoplastic population of case no. 8, while beingHLA-DR-positive (Fig. 4.2), lacked HLA-DQ. In case no. 5 themajority of tumour cells reacted with 2 06 (Fig. 2.2), TU22,TUY34, thus indicating the presence of HLA-DQ and - DRmolecules on their surface. Leu 10, however, (which is restrictedin binding to the HLA-DQwl and w3 alleles) (Brodsky, 1984)stained only a very small proportion of tumour cells. Thissuggests that the patient expressed either DQwl or - DQw3 (orboth) and had tumour cells defective for this particular allele.

Mab VIC-Yl directed against the HLA-D associated invari-ant chain gave rise to an exclusively cytoplasmic binding pattern(Fig. 2.3). The VIC-Y1 and anti-DR mAb gave similar stainingpatterns in all seven cases. In case no. 5 a small subset of theneoplastic cell population contained Ii (Fig. 2.3) while it lackedHLA-DR (Fig. 2.2) and - DQ. In case no. 8, in which thetumour cells failed to express -DQ antigens, there was uniformIi expression.

Taken together, this series of Ig-deficient mature B-celllymphomas contains tumours with variable defects in MHCantigen equipment; three lacked both class I and class IIantigens; four other tumours contained varying proportions ofMHC antigen-deficient neoplastic cells and there was coordi-nate expression ofMHC class I, HLA-DR and -DQ antigensin only one case.

DISCUSSION

It has been shown that the expression of HLA antigens isdynamic. It depends on inductive/enhancing stimuli such as

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Figure 2. Case no. 5 (2.1) Reactivity ofmAb W6/32 is clearly restricted tothe connective tissue cells and to very few scattered histiocytic cellsindicating the lack of expression of HLA-A,B,C on the neoplasticpopulation (AEC/H; x 84). (2.2) Ti22 (anti-HLA-DQ)-reactivityresults in an intense staining of dendritic interstitial cells and scatteredhistiocytes and in a faint surface staining of a considerable proportion ofneoplastic cells while clearly unstained tumour cells are also present(e.g., compare square 1 and 2) (AEC/H; x 133). (2.3) Demonstration ofthe HLA-D associated invariant chain (Ii) with mAb VIC-Y1. Invariable amounts the large blastic tumour cells contain 1i in theircytoplasm. The small Ii-negative intermingled lymphocytes are reactiveT cells (data not shown) (AEC/H; x 133).

interferons and tumour necrosis factor (Rhodes, Jones &Bleehen, 1983; Rosa & Fellous, 1984; Nissen et al., 1985;Berman et al., 1985; Collins et al., 1986) and inhibitory stimulisuch as corticosteroids (Hokland et al., 1981) and prostaglandinE2 (Berman et al., 1985; Snyder, Beller & Unanue, 1981). Onnormal B cells and under physiological conditions, however,class I antigens are constantly exposed throughout the matu-ration (Maloy & Coligan, 1985; Alexanian, Barlogie & Fritsche,1985). Class II molecules appear at the stem-cell level, increasein density after binding of antigen to surface Ig (Cambier et al.,1985) and decline in expression at the mature plasma-cell level(reviewed by Guy et al., 1986b). B lymphocytes express at leastthree HLA-D-region molecules, - DP, - DR, and - DQ(Hurley, Giles & Capra, 1983) for which a sequential geneexpression in this order has been suggested (Guy & vanHeyningen, 1983). Each of these subregions controls a distincta- and f-chain, that are intracellularly non-covalently asso-ciated with a non-MHC, non-polymorphic 'invariant' chain (Ii)(Owen et al., 1981).

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Figure 3. Case no. 6 (3.1) This tumour contains a small percentage of large blastic W6/32-reactive probably histiocytic cells although it cannot beexcluded that they belong to the tumour cell population that otherwise lacks detectable class I molecules (AEC/H; x 133). (3.2) The class II frameworkmAb 2-06 stains endothelial and interstitial dendritic cells; the HLA-D-negative neoplastic B-cell population contains few scattered weakly stainedblastic cells of uncertain origin (AEC/H; x 133).

Congenital defects in MHC antigen expression on B cells areknown to exist in primary combined immunodeficiency, where adefect in HLA-A and -B antigen expression (Schuurmann et al.,1979) and a defect in class II gene expression (Lisowska-Grospierre et al., 1985) could be observed, while intracellular Ii-chain levels were normal.

On the other hand, there are reports on MHC-antigendefects on neoplastic B-cells. The Daudi line has defects in 2-microglobulin expression (Rosa et al., 1983). The RJ 2.2.5variant of the Raji cell line, although equipped with intact classII genes, is devoid of class II gene products, probably due to arepression of transcription (Long et al., 1984; Accolla, Carra &Guardiola, 1985). P3HR-1 is another B-lymphoid cell line

known to be defective for class II antigen expression (Trow-bridge, Hyman & Klein, 1977). A selective non-expression ofHLA-DQ was found on the B-cell lines Josh 7, Reh, Nalm 12using mAb Leu 10 (Wang et al., 1983).

Data on in situ demonstration of defects in class I antigenexpression in malignant lymphomas are scarce. Woda, Rackin& Rappaport (1981) demonstrated absence of HLA-A,B,C-molecules and #2-microglobulin but presence of HLA-DRantigens in two cases of large, non-cleaved 'histiocytic' lym-phoma. The corresponding constellation is given in our case no.5. Class II antigen defects in B-cell lymphomas were sporadi-cally found by several authors (Barcos et al., 1983; Pallesen etal., 1984; Anderson et al., 1984; Knowles II et al., 1985). In a

.?.- _ is4.:

Figure 4. Case no. 8 (4. 1) mAb B9. 12.1 fails to react with large amounts of the neoplastic population while interstitial dendritic cells, fibrocytes andscattered histiocytes are intensely labelled, thus indicating a partial loss of class I expression (AEC/H; x 133). (4.2) In contrast to the partial defect inclass I antigen- and HLA-DQ-expression, this tumour is entirely HLA-DR positive as demonstrated by reactivity ofmAbs 2-06 and Tf134. Note theunreactive connective tissue fibres and a vascular lumen (AEC/H; x 133).

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series of 20 B-cell lymphomas, Krajewski et al. (1985) detected aselective lack of HLA-DQ antigen expression in several casesincluding two diffuse lymphocytic and one centrocytic lympho-ma(s). This selective defect was found again in a larger seriesanalysed by Guy et al. (1986b) who coined the term 'non-coordinate expression' for it. However, this phenomenon wasnot new at that time: Swerdlow et al. (1984) reported that theanti-DR mAbs Da-2 and Ca-2.11 recognized at least in severalcases more lymphoma cells than did mAb 33- 1, a putative - DQreagent. LeulO was used by Al-Katib & Koziner (1983) to detectdifferences in - DR and - DQ expression on 28 cases of B-cellchronic lymphocytic leukaemia, the neoplastic cells, four ofwhich failed to react with this mAb. Faille, Turmel & Charron(1984) contributed a case of hairy cell leukaemia lackingexpression of - DQ molecules in the presence of - DR; this lackcould be restored after short term culture in presence ofphorbolester. We could demonstrate the selective -DQ antigen defi-ciency once, in case no. 8.

Case no. 5 contained a subset of neoplastic cells thatexpressed Ii although being devoid of HLA-DR and - DQ. Inthe mouse Ta-/Ii+ lymphocytes have been recently described(Momburg et al., 1986). However, for human lymphocytes thisdissociation has not been reported so far. Thus, our observationwill have to be confirmed in a larger series of B-cell lymphomas.

On this background the complete lack ofMHC antigens incases no. 3, 7, and probably 6, the isolated lack of class Imolecules in case no. 5 and the severe defects in expression ofclass I and II molecules in cases no. 1, 4, and 8 are striking andseem to be in line with one principal characteristic of thislymphoma group: its complete loss of Ig-expression. Since classII molecules are transducers of signals for B-cell activation(Bonagura et al., 1985; Corley et al., 1985; Clement, Tedder &Gartland, 1986) and maturation (Palacios et al., 1983), a lack ofthese important trigger molecules probably contributes to theincomplete differentiation of the neoplastic B cell. Class Iantigen-deficient tumour cells possibly escape immune attack bycytotoxic T cells (Sanderson & Beverley, 1983). For coloncarcinoma non-expression of class I antigens could be related toa poorer degree of differentiation (Momburg et al., 1986) thatitself is correlated with unfavourable prognosis. These argu-ments are in line with the unfavourable clinical course of thisspecial lymphoma group.

ACKNOWLEDGMENTS

This work was supported by the Tumorzentrum Heidelberg/Mannheimand the Land Baden Wurttemberg (Forschungsschwerpunktprogramm17-9). We are indebted to Dr Harry Schmitteckert from the LVA-Klinikfur Thoraxerkrankungen (Heidelberg/Rohrbach) for supplying clinicaldata and to Ms I. Brandt and to Mr J. Moyers for their skillful technicalassistance.

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