Lymphocyte populations and TAC-antigen in diffuse B-cell lymphomas

Leukemia Research Vol. 10, No. 11, pp. 1271-1278, 1986. Printed in Great Britain.

0145-2126/86 $3.00 + .00 (~ 1986 Pergamon Journals Ltd.

LYMPHOCYTE POPULATIONS AND TAC-ANTIGEN IN DIFFUSE B-CELL LYMPHOMAS* BARBARA W. GRANT, JEFFREY L. PLATT, HARRY S. JACOB and NEIL E. KAY Department of Medicine, Section of Hematology-Oncology, Minneapolis Veterans Administration Medical Center, and Department of Pediatrics, Division of Nephrology, and Department of Medicine, Section of Hematology, University of Minnesota Hospital, Minneapolis, MN, U.S.A. (Received 11 February 1986) Abstract Immune cell populations in 8 diffuse histology B-cell lymphomas were analysed in frozen tissue sections by indirect immunofluorescence to gain insight into their possible modulating influence in these tumors. Use of monoclonal antibodies to identify cellular and extracellular antigens combined with nuclear counterstaining allowed precise quantitation, localization and comparison of T- and B-lymphocyte populations. T lymphocytes clustered in non-random fashion. Areas of high T-lymphocyte density manifested higher T4:T8 ratios than locales with fewer T lymphocytes (p < 0.05). Few cells bad surface antigens (Leu 7, 73.1, OKM1) associated with natural killing. Cells strongly reactive with anti-TAC (Interleukin-2 receptor, associated with Tlymphocyte activation) were also T l l reactive and were usually helper (Leu 3) phenotype. In addition, B-lineage lymphoma cells in some tissues reacted with anti-TAC. The pattern of tumor cell reactivity with anti-TAC correlated with Rappaport histologic classification. These findings suggest that non-malignant T lymphocytes modulate B-lymphoma cell growth in situ, and that in some lymphomas the T-cell product IL-2 may be an important local growth factor. Key words: Lymphoma, IL-2 receptor, T lymphocytes.

INTRODUCTION IMMUNOHISTOCHEMICALanalysis of human non-Hodgkin's lymphomas suggest these tumors are clonal expansions of single malignant B cells since most tumor cells express a single immunoglobulin light chain type [1-5] or idiotype [6]. However, evaluation of suspensions of malignant B-cell nodes consistently reveals a population of cells with T-lymphocyte surface antigen characteristics [1-5]. Monoclonal antibodies can be used to identify peripheral blood T-lymphocyte phenotypes which correlate closely with functional roles in controlling B-lymphocyte proliferation and differentiation [7]. Monoclonal antibodies have also been used to characterize the distribution of T-lymphocyte phenotypes in * This work was supported in part by funds from the Minnesota Medical Foundation and the Merit Review of the Veterans Administration and by grants NIH AI-10704, AM25518, Am26149, and HL07062. JLP is under tenure of a Clinical Scientist Award from the American Heart Association. Presented in part at the annual meeting of the American Society of Hematology, San Francisco, December 1983. Abbreviations: IL-2, interleukin-2; FITC, fluorescein isothiocyanate; TRIC, tetraethylrhodamine isothiocyanate; LCM, leukocyte conditioned medium. Correspondence to: Dr Barbara W. Grant, Section of Hematology, Given Building C-312, University of Vermont, Burlington, VT 05405, U.S.A.

normal lymphoid tissue [8--10], renal graft rejection [11], graft vs-host disease of skin [12], delayed type hypersensitivity [13] and nodular lymphomas. We have intensively studied lymph nodes from 8 patients with diffuse B-cell lymphomas using monoclonal antibodies by indirect immunofluorescence to locate, characterize, and quantitate both tumor cells and associated T lymphocytes in situ. Our investigation demonstrates that: (1) T lymphocytes associated with diffuse B-cell lymphomas reside in T-cell "rich" or Tcell "poor" extravascular locales wherein distinct subpopulations of T lymphocytes predominate; (2) few cells in these tumors have surface antigens associated with natural killing function of blood and bone marrow cells; (3) B-lineage lymphoma cells in some diffuse lymphomas bind anti-TAC, a monolconal antibody which recognizes interleukin-2 (IL-2) receptors on activated T lymphocytes [14-16]. MATERIALS

AND METHODS

Source of lymph nodes Eight consecutive tissues obtained from patients with diffuse B-cell lymphoma and three normal lymph nodes were studied. Patients had undergone lymph node resection for diagnostic purposes, and had given informed consent to this protocol which was approved by the Human Subjects Committee at the Minneapolis V.A. Medical Center. These eight patients were

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male, aged 49-76. Each had stage IV disease except patient 6 who had stage III. One patient (No. 5), who was in relapse, had previously received treatment with combinations of Adriamycin, Bleomycin, Cytoxan, Prednisone and Vincristine for a total of 20 months. Six additional similar diffuse lymphoma tissues were studied after reaction with BA-1, anti-TAC and T l l only. Histologic diagnoses were made by the clinical laboratory on sections from each node. Two to four cubic millimeter portions of nodes were snap frozen in precooled isopentane and stored at -70°C until use.

Monoclonal antibodies The monoclonal antibodies used in this study are described in Table 1. Preparation and staining of node sections Four-micron thick frozen sections were prepared in a Lipshaw Cryostat, air dried, acetone fixed, and washed with phosphate buffered saline, pH 7.4 as previously described [11]. Sections were sequentially reacted with appropriate dilutions of monoclonal antibodies. F(ab')2 fluorescein isothiocyanate (FITC) conjugated rabbit antimouse IgG (heavy and light chains) (Zymed Laboratories, San Francisco), F(ab')2 FITC goat antirabbit IgG (heavy and light chains) (Tago, Burlingame, CA), and p-phenylenediamine to retard fluorescence fading [17]. Ethidium bromide was used in some preparations to stain nuclei [II]. In addition, sections of each tissue were reacted with monospecific F(ab')2 FITC goat antihuman IgG(6) and IgM(~t) (TAGO). Tissue analysis Sections were examined at 500 x with a Zeiss Universal microscope equipped for epifluorescence with a 10 x 10ram ocular indexing grid. Regions of lymph nodes having relatively high and low T-lymphocyte density (referred to as "T-rich" and "T-poor", respectively) were located on sections reacted with T l l . These regions were then identified on serial sections reacted with other monoclonal antibodies and the following were enumerated: (1) total lymph node cells identified by orange-red nuclear reactivity with ethidium bromide and (2)

reactive cells having an apple-green fluorescent plasma membrane surrounding an orange-red nucleus. Autofluorescent and enucleated cells were excluded. At least 1000 lymph node cells in each selected T-rich and T-poor locale in each tissue section were enumerated. Analysis of different areas of the same section or of different sections of the same node yielded consistent results. Results were expressed as percent of total cells. Data were analysed using the two-sample rank test.

Localization of reactive cells To precisely locate T lymphocytes with respect to vasculature, sections of each tissue were sequentially reacted with T l l or OKT8; MBM4 (which identifies Type IV collagen in vascular basement membranes) [18], and a double fluorochrome layer as described above. In some studies tetraethylrhodamine isothiocyanate (TRIC) conjugated rabbit antihuman basement membrane antibody [19] was applied to sections which had been reacted with OKT8 FITC or T l l FITC. Dual labeling methods Analysis of pairs of antigens in a given tissue was accomplished using two different monoclonal antibodies applied as a mixture or in series as follows: (1) the binding of IgM monoclonal antibodies BA-1 and Leu 7 was compared with that of IgG monoclonal antibodies T l l , OKT8 and antiTAC using FITC F(ab')2 goat antimouse IgM (Cappel Laboratories, Inc., West Chester, PA) and TRIC goat antimouse IgG (Fc) (Cappel); (2) anti-TAC (IgG2a) and T l l (IgG1) binding were compared by sequential application of anti-TAC, rabbit antimouse IgG2~ (Litton Bionetics, Inc., Kensington, MD), mouse serum absorbed TRIC (F(ab')2 goat antirabbit IgG (heavy and light chains) (Cappel) and T l l FITC; (3) antiTAC and OKT8 reactivity were compared by sequentially reacting tissue with anti-TAC, TRIC goat antimouse IgG (Fc), mouse serum and OKT8 FITC; (4) anti-TAC and Leu 3a + 3b binding were compared by sequential application of anti-TAC, TRIC F(ab')2 rabbit antimouse IgG (heavy and light chains) (Cappel), mouse serum and FITC Leu 3a + 3b (Bectin-Dickinson); (5) T l l (IgG0 and OKT8 (IgG2a) were compared

TABLE 1. MONOCLONALANTIBODIES Antibody

Mouse Ig subclass

T l l and T l l FITC OKT4 Leu-3a, 3b FITIC OKT8 and OKT8 FITC OKM1 Leu-7 73.1 OKT9 Anti-TAC BA-1

IgG~ IgG2b IgGza IgG:a IgG2b IgM IgG2~ IgG1 IgG2~ IgM

BA-2 MBM4

IgG3 IgG2b

Specificity and source T lymphocytes, Coulter Immunology, Hialeah, FL. Helper T lymphocytes, Ortho Pharmaceuticals, Inc., Raritan, NJ. Helper T lymphocytes, Bectin-Dickinson, Mountain View, CA. Cytotoxic/suppressor T-lymphocytes, Ortho. Monocytes, granulocytes and natural killer cells, Ortho. Large granular lymphocytes Bectin-Dickinson. Natural killer cells, G. Trinchieri, Wistar Institute, Philadelphia, PA. Transferrin receptor, Ortho. Interleukin-2 receptor, T. Waldmann, Bethesda, MD. Mature B lymphocytes and PMNs, T. LeBien, Univ. of Minnesota, Minneapolis, MN. P24 leukemic cell antigen, T. LeBien. Type IV collagen, A. F. Michael, Univ. of Minnesota.

9 e

FIG. 1. Diffuse B-cell lymphoma reactivity with monoclonal antibodies as demonstrated by indirect immunofluorescence on frozen tissue sections. (a) Diffuse BA-1 reactivity of WDLL (500x). (b) A PDLL tissue is diffusely reactive with BA-2. Fluorescence intensity varies focally (275x). (c) A WDLL tissue reacted with both T11 and MBM4 (type IV collagen of vascular basement membrane). Most T l l reactive cells are parenchymal. A single T cell (arrow) is present within a blood vessel (arrow heads) (500x). (d) T l l reactive cells in a WDLL tissue are distributed in areas of relatively high ("T-rich") and low ("T-poor') density (275 x).

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FIO. 2. Anti-TAC reactivity of WDLL demonstrated by indirect immunofluorescence on frozen tissue section. A strongly reactive T lymphocyte is seen in the midst of less fluorescent B-lymphoma cells (50x).

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Lymphocyte populations in B-cell lymphomas by sequential application of OKT8, rabbit antimouse IgG2a, mouse serum absorbed TRIC F(ab')~ goat antirabbit IgG (heavy and light chains) and T l l FITC. Standardization o f monoclonal antibodies and controls

Monoclonal antibodies (except anti-TAC) were standardized by immunofluorescence using frozen sections of normal lymphoid tissues [8-11]. Anfi-TAC was standardized as follows: the maximal dilution with undiminished fluorescence of anti-TAC ascites (1:40,000) was ascertained by indirect immunofluorescence on frozen tissue sections. Anti-TAC at 1:20,000 and 1:40,000 dilutions was then adsorbed for I h at 25°C with either stimulated or unstimulated T lymphocytes which had been prepared as described below and then washed three times in RPMI. Stimulated T lymphocytes were obtained by incubation of E-rosette isolated T lymphocytes in leukocyte conditioned medium (LCM) at 37°C for 4 days. LCM was prepared by incubation of peripheral blood mononuclear cells in Iscove's Modified Delbecco's Medium (Gibco Labs, Grand Island, NY), 15% fetal calf serum (Reheis Chemical Company, Phoenix, AZ), and 1% phytohemagglutinin (Burroughs Wellcome, Triangle Park, NC) for 4 days. Unstimulated T lymphocytes were obtained by the E-rosette method. Frozen sections of three tissues known to contain numerous anti-TAC binding cells were then reacted with one of the absorbed antiTAC antibody solutions followed by a double fluorochrome layer as described above. Absorption of anti-TAC with stimulated but not with unstimulated T lymphocytes abrogated tissue fluorescence. Anti-TAC identified occasional cells in the medullary cords and interfollicular areas of normal lymph nodes, while rare (<1 per follicle) anti-TAC reactive cells were seen in B-rich follicles and germinal centres. A similar pattern of anti-TAC reactivity in normal nodes has been described by Miyawaki et el.

[2o1.

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All heteroantisera were immunoabsorbed with human plasma. Each study was controlled by preparation of tissue sections as described above but omitting application of the monoclonal or of one or another of each pair of monolconal antibodies. In addition, double labeling studies were controlled using appropriate tissue targets such as NZB/NZW mouse kidney to exclude cross reactivity of antisera.

RESULTS Each tumor studied was diffusely reactwe with BA1 [21], and all except tissue No. 7 expressed surface immunoglobulin (IgD or IgM). Reactivity with BA-2 [22] however, varied considerably and did not correlate with tumor histology (Table 2; Figs la, b). Cells reactive with T l l were distributed throughout the nodal specimens. To exclude tumor cell binding of T l l , sections of each tissue were reacted with a mixture of T l l and BA-1 followed by FITC antimouse IgM and TRIC antimouse IgG as described above. These studies revealed that less than 1% of T l l reactive cells were also reactive BA-1. OKT8 reactive cells also failed to react with BA-1, while studies using T l l and OKT8 together on node sections as described above demonstrated that OKT8 reactive cells were also T11 reactive. To precisely locate T lymphocytes with respect to nodal vasculature, sections of each study tissue were reacted sequentially with T l l and MBM4 (type IV collagen, vascular basement membranes) followed by a double fluorochrome layer as described above, and with FITC T l l and TRIC rabbit antihuman basement membrane antibodies [19]. These studies demonstrated that T lymphocytes largely resided in extravascular locales (Fig. lc).

TABLE 2.

C O R R E L A T I O N OF DIFFUSE B-CELL LYMPHOMA HISTOLOGY WITH MONOCLONAL ANTIBODY BINDING DETECTED BY INDIRECT IMMUNOFLUORESCENCE IN FROZEN TISSUE SECTIONS

Tumor cell reactivity with monoclonal antibodies Patient No.

Rappaport histology*

BA-2

OKT9

anti-TAC

l 2 3 4

WDLL WDLL WDLL IDLL

Diffuse Focal aggregates Negative Occasional cells

Focal aggregates Negative Occasional Focal aggregates; variable intensity

5

PDLL

6

PDLL

Diffuse; variable intensity Diffuse

Diffuse; variable intensity Occasional cells

7 8

DHL DHL

Occasional cells Occasional rare cells

Diffuse Focal aggregates

Diffuse Diffuse Diffuse Diffuse; variable intensity Focal aggregates Focal aggregates Negative Negative

All patients were male, with stage III or IV disease, and all tumors reacted brightly and diffusely with BA-1. * WDLL, well differentiated lymphocytic lymphoma; IDLL, intermediately differentiated lymphocytic lymphoma; PDLL, poorly differentiated lymphocytic lymphoma; DHL, diffuse histiocytic lymphoma.

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In seven of eight tissues T lymphocytes were nonrandomly distributed such that T-rich and T-poor areas could be easily discerned (Fig. ld). The percentage of T lymphocytes in the T-rich and T-poor areas varied from node to node (Table 3), but was consistent for a given tissue. In one tissue (patient 3) T l l reactive cells were randomly distributed. Analysis of sequential sections of tumor specimens revealed that OKT4 r e a c tive cells were generally more plentiful than OKT8 reactive cells in the T-rich areas, while in T-poor areas OKT4 often bound fewer cells than did OKT8 (Table 3). The T4: T8 ratio in T-rich areas exceeded that in Tpoor areas (p < 0.05). Tumor cell reactivity with OKT9 [23, 24] and BA-2 was not different in T-rich or T-poor areas. Monoclonal antibodies which recognize peripheral blood lymphocytes having natural killer function identified few cells in the malignant nodes. Leu-7 reacted with 0.1-6% of nodal cells which were distributed similarly to T l l reactive cells. OKM1 [25] and 73.1 [26] identified rare mononuclear ceils in these nodes (0-0.5%). A variable number of cells strongly fluorescent with anti-TAC were seen in all lymphoma nodes. Doublelabeling studies demonstrated that these cells were reactive with T l l , but not with BA-1 and that the proportion of cells reactive with both T l l and anti-TAC was similar in T-rich and T-poor loci. Most cells strongly reactive with anti-TAC were also reactive with Leu-3a, 3b (analogous to OKT4), while fewer than 1% reacted with OKT8. A n t i - T A C reactive cells, however, were often observed adjacent to OKT8 reactive cells. A second population of cells less strongly fluorescent with anti-TAC (Fig. 2) was observed in six of eight lymphoma nodes. These cells were identified as tumor cells by nuclear morphology, distribution, and by double-labeling studies using anti-TAC and BA-1.

TABLE 3. T-CELL DISTRIBUTION AND PHENOTYPES WITHIN DIFFUSE HISTOLOGY B-CELL LYMPHOMAS IDENTIFIED BY INDIRECT IMMUNOFLUORESCENCE IN FROZEN TISSUE SECTIONS

Percent T l l reactive Tissue No. 1 2 3 4 5 6 7 8

OKT4 : T8 ratiot

T-Rich* areas

T-Poor* areas

T-Rich* areas

T-Poor* areas

31 32 7 21 23 36 10 55

6 16 7 13.6 9 3 3 22

0.85 3.8 5 1.5 2.08 1.67 7.7 1.14

0.5 1.4 5 0.88 0.79 0.65 1 0.41

* T-Rich and T-Poor refer to areas identified on T11 reacted sections which had high or low densities of T lymphocytes, respectively. t Ratio of percent of lymph node ceils in specified area reactive with monoclonal antibody OKT4 to percent reactive with OKT8. The OKT4:T8 ratios in T-rich areas exceed that in T-poor areas (p < 0.05 by two sample rank test).

Binding of anti-TAC in these tissues correlated with tumor histology (Table 3), and this was confirmed in six additional diffuse B-lymphoma tissues. Of the fourteen tissues studied with anti-TAC, anti-TAC reacted with all BA-1 reactive tumor cells in each of four well-differentiated lymphocytic lymphoma (WDLL) nodes, and one of two intermediately differentiated lymphocytic lymphoma (IDLL) nodes, as well as focal areas of tumor cells in the other I D L L and three of three poorly differentiated lymphocytic lymphoma (PDLL) tissues. In contrast anti-TAC did not bind BA-1 reactive tumor cells in any of five diffuse histiocytic lymphoma tissues, though in these tissues it did identify a population of T lymphocytes similar to that observed in WDLL, IDLL and P D L L nodes.

DISCUSSION T lymphocytes have the capacity to regulate normal B-lymphocyte proliferation and differentiation [7] and to kill transformed lymphoblastoid cells in vitro [27]. Although T lymphocytes are consistently found admixed with malignant B lymphocytes when cell suspensions of lymphomas are studied [8-10, 15], the influences of these T-phenotype cells in the biologic behavior of the malignant cells is not known. Monoclonal antibodies which identify functionally defined lymphoid populations have been used to characterize the distribution of lymphoid cells in normal lymph nodes [9] and to suggest phenotypic-functional associations [8, 10]. We have examined the distribution and phentoypic markers of lymphoid cells in diffuse B-cell lymphomas using monoclonal antibody probes and indirect immunofluorescence. Our studies demonstrated that: (1) T l l reactive cells are clearly not BA-1 reactive, and presumably are not B-lineage lymphoma cells; (2) nodal T lymphocytes cluster in non-random fashion in extravascular locales, T-rich areas having higher T4 : T8 ratios than T-poor areas; (3) few mononuclear cells with phenotypes associated with natural killer function (Leu7, OKM1, 73.1) are present; and (4) anti-TAC which reacts with the IL-2 receptor on activated T lymphocytes [14, 16] also binds B-lineage ( B A - I + ) tumor cells in patterns predictable by Rappaport classification. Both normal nodes and follicular lymphoma nodes contain abundant T lymphocytes at the periphery of B-rich follicles, but few within follicles [9, 10, 28, 29]. Quantitative studies reveal that the T4:T8 ratio in the perifollicular area is high (about 3:1), and that the sparse intrafollicular T cells are almost exclusively T4+ T 8 - [9, 10, 28]. The T-cell percentages and T4:T8 ratios in the T-rich areas of the diffuse histology nodes we studied were similar to those described in interfollicular areas of nodular lymphomas. However, in Tpoor areas of diffuse histology nodes T4: T8 ratios were consistently lower than those reported in any areas of follicular lymphomas or normal nodes [9, 10, 28]. Whether relative enrichment of OKT8 reactive cells in these T-poor loci reflect host attempts at immunomodulation or tumor suppression is not known. Relative enrichment for OKT8 reactive cells has been observed

Lymphocyte populations in B-cell lymphomas in follicular and interfollicular area of nodes from patients with acquired immunodeficiency syndrome [18, 30]. Most studies of T-B lymphocyte interaction have shown that OKT4 or Leu-3 reactive cells enhance Bcell proliferation [7], and that they are the major source of the lymphocytic growth factors BCGF and IL-2. In addition, however, some OKT4 reactive cells recognize and are cytotoxic for cells with allogeneic Class II major histocompatibility antigens [31, 32] or self plus viral antigens [33]. The role of OKT4 reactive cells in enhancement or control of B-cell tumor growth, however, is unknown. Their potential importance is suggested by our observation that anti-TAC reactivity was confined to the Leu-3 reactive T-lymphocyte subpopulation. The monoclonal antibody anti-TAC which reacts with interleukin-2 receptors expressed by activated or proliferating T lymphocytes [14--16], has been shown here to bind with BA-1 reactive lymphoma cells in a manner predictable by tumor histology. Anti-TAC has recently been described to react with activated B lymphocytes [34-36] as well as with hairy cell leukemia cells which have rearranged immunoglobulin genes consistent with B lineage [37]. Our findings suggest that WDLL and some IDLL lymphoma cells represent stages of B-lymphocyte development during which IL-2 receptors are expressed, It will be important to determine whether these cells are dependent on the T-cell product IL-2 for growth, and if so whether they get it from nodal T lymphocytes, or can synthesize it themselves. Immunohistochemistry allows in-situ analysis of antigen distribution and avoids potential selection problems inherent in studying node cell suspensions. As much as membrane antigens can be correlated with function, such studies provide an initial approach for assessing invivo tumor cell and host lymphocyte micro-environment. While it is still unclear why T lymphocytes are found so ubiquitously in diffuse B-cell neoplasmas, the present study suggest that they likely play a role in the growth or suppression of the neoplastic cells.

Acknowledgements--We wish to thank Dr Thomas Waldmann for generously providing anti-TAC; Dr Tucker LeBien for BA-1 and BA-2; Dr Giorgio Trinchieri for 73.1; Ms Kathy Devine and Mr Marshall Hoff for technical assistance.

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