Thy-1 epitopes are expressed on murine myeloid leukemia and reticulum sarcoma cells

Immunology Letters, 6 (1983) 187-190

Elsevier BiomedicalPress

THY-1 EPITOPES A R E E X P R E S S E D ON M U R I N E M Y E L O I D L E U K E M I A A N D R E T I C U L U M S A R C O M A CELLS J. BUBEN]K, D. BUBEN]KOVA*, T. JANDLOVA, R. HOLUSA* and V. SVOBODA* Institute of Molecular Genetics, Czechoslovak Academ.r oJ' Sciences. 166 37 Prague 6 and *Institute q[' l~vgiene and Epidemiolog.r, I00 42 Prague I0. Czechoslovakia

(Received 17 November 1982) (Modified versionreceived31 January 1983) (Accepted I February 1983)

1. Summary Expression of Thy-l.2 specificities in cells from 29 primary spontaneous leukemias of random-bred 1CR Swiss mice was examined by cell membrane and cytoplasmic immunofluorescence with monoclonal HO-13-4 antibody [1]. The Thy-l.2 epitopes were detected in all thymic lymphomas and were absent in the lymphomas of non-thymic origin. Unexpectedly, the Thy-1.2 epitopes were also detected in 71% (5/7) of myeloid leukemias and 40% (4/10) of reticulum cell sarcomas examined.

2. Introduction The Thy-I antigen, described first by Reif and Allen [2], has been shown to be present on cells of T-cell lineage in two allelic forms, Thy-l. 1 and Thy1.2; these determinants are believed to discriminate between thymus-derived lymphocytes and other lymphohematopoietic cells [3]. The Thy-I antigen has an interesting tissue distribution. It has been found to be present on neural cells [2,4], epidermal cells [4], fibroblasts [5], normal and neoplastic mammary tissue [6,7] and transitorily in skeletal muscle development [8]. Recently the Thy-I epitopes were found to be expressed on nonmalignant murine hematopoietic cells other than Tcells [9,10]. Key words: Thy-I antigen - immunofluorescence monoclonal antibodies reticulumcell sarcomas myeloidleukemias

Using monoclonal Thy-l.2 antibodies we report here that Thy-1 epitopes can also be detected on murine reticulum cell sarcomas and myeloid leukemia cells.

3. Materials and methods 3.1. M i c e

Females from a closed colony of random-bred ICR Swiss mice (VELAZ, Czechoslovakia) with high incidence of spontaneous neoplastic diseases of hematopoietic and lymphoid tissues [1 I] were used in this study. 3.2. L e u k e m i a s Twenty-nine primary leukemias, which originated in 154-574-day-old ICR Swiss mice, were examined. Mice with enlarged lymph nodes and/or spleens were killed when moribund. The classification of the leukemias [12 14] was based on autopsy, histology (lungs, spleen, liver, thymus, enlarged lymph nodes, kidneys, bone marrow and eventually other tissues were routinely examined) and cytological typing of the neoplastic diseases performed independently by two pathologists. Each leukemia was examined by immunofluorescence. However, the results of the immunofluorescence tests were included in this paper only when the classification of the leukemia given by both pathologists was identical. Portions of the same enlarged (usually more than 10 mm in diameter) mesenteric or axillary lymph node were used

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for simultaneous histological, cytological, immunofluorescence and other [ 15,16] examination. 3.3. hnmunofluorescence Monoclonal Thy-l.2 antibody produced by HO13-4 hybridoma [1] was kindly provided by Dr. L. Steiner, Massachussetts Institute of Technology, Boston, MA, U.S.A. The HO-13-4 monoclonal product is 19S lgM with a specific lytic titer higher than 10 6 in a 51Cr release assay. Cytotoxicity, immunofluorescence (IF), cell sorting and specific elimination of cytotoxic T-lymphocytes, their precursors and Con A-responding cells after treatment with HO-13-4 antibody indicated strict Thy-l.2 specificity of the antibody [l]. The above data were confirmed in this laboratory (Indrovfi and Bubenlk, unpublished observations). The HO-13-4 antibody preparation diluted to contain 2.5 mg IgM/ml was kindly conjugated with fluorescein isothiocyanate (FITC) by Dr. P. ManUal, SEVAC, Prague. For staining of cell membranes in suspensions of leukemic cells and for cytoplasmic immunofluorescence performed with acetone-fixed cell smears, 50 ~zg/ml FITC-HO-13-4 Thy-l.2 antibody and 5 × 106 cells were used. The cells were processed and the immunofluorescence was evaluated as described earlier [17].

4. Results

Among 29 murine leukemias examined, 9 were diagnosed as generalized thymic lymphomas or lymphosarcomas, 3 as non-thymic lymphomas, 10 as reticulum cell sarcomas and 7 as myeloid leukemias. In all lymph nodes used for immunofluorescence examination the original structure was obliterated and completely replaced by neoplastic cells. From each lymph node a cell suspension was prepared and examined by membrane immunofluorescence with FITC-HO-13-4 Thy-l.2 monoclonal antibody. Simultaneously, acetone-fixed cell smears were examined by cytoplasmic immunofluorescence with the same preparation and dilution of the FITC-HO-I 3-4 antibody. The results obtained with representative leukemias of various histological types are shown in detail in Table !; the results of 1F tests performed with all leukemias examined are summarized in Table 2. In general, there was a good concordance be188

Table 1 Thy- 1.2 epitopes detected by FITC-labeled H O- 13-4 monoclonal antibody in mouse leukemias Leukemia

Percentage of IF + cells Membrane IFa

Thymic lymphoma T3 T5 -I"6

42.9 75.2 73.1

Cytoplasmic I P

40.0 89. I 92.3

Non-thymic lymphoma LI L2 L3

0 0 0

Reticulum cell sarcoma R3 R8 R9

29.3 49,9 71,8

35.5 56.3 100.0

Myeloid leukemia MI M2 M3

41.3 64.2 76.6

45.0 70.6 98.1

0 0 0

a 150-250 cells were examined in each cell sample; briUiant green immunofluorescence (ring-reactions, caps or sectorial membrane fluorescence) was considered positive. b 2 5 acetone-fixed cell smears prepared from fresh suspension of each leukemia or stored at -20 ° C for 2-4 months were examined. The percentage of cells displaying diffuse cytoplasmic fluorescence was based on examination of 100 ceils in each cell smear; mean values of 2 5 cell smears are given in Table I. The intensity of immunofluorescence in 1F+ cells of thymic lymphomas, reticulum cell sarcomas and myeloid leukemias was comparable.

Table 2 Percentage of Thy-l.2 + leukemias occurring spontaneously in ICR Swiss micea Leukemias

No. of Thy- 1.2+ leukemias/Total no. of leukemias examined (%)

Thymic lymphomas Non-thymic lymphomas Myeloid leukemias Reticulum cell sarcomas

9/9 (100) 0/3 (0) 5/7 (71) 4/10 (40)

a The criteria of immunofluorescence-positivity are given in the legend to Table 1; leukernias containing more than 20% of IF + cells were considered Thy-1.2 +.

tween the results obtained with cell-surface and cytoplasmic fluorescence. The cytoplasmic fluorescence showed in most cases a slightly higher percentage of Thy-l.2 + cells (Table 1). The Thy-l.2 antibodies discriminated reproducibly between lymphomas of thymic and non-thymic origin (Tables 1 and 2). Unexpectedly, 71% (5/7) of myeloid leukemias and 40% (4/10) of reticulum cell sarcomas were found to be Thy-l.2 + (Table 2). The percentage of Thy-l.2 + cells in myeloid leukemias and reticulum cell sarcomas was comparable with that of thymic lymphomas (Table 1).

myeloid leukemias. The Thy-l myeloid leukemias and reticulum cell sarcomas can either be considered as originating from Thy-I precursors, or they may originate from Thy-I + precursors with quantitatively decreased content of the Thy-I antigen. Thy-l antigen can also be lost or quantitatively decreased during the process of malignant conversion of the precursor cells or during further progression of the malignantly converted cell population, Whatever the interpretation, our results support the view that Thy-I can no longer be regarded as an exclusive marker for normal and malignant cells of the T-cell lineage.

5. Discussion Until recently it was believed that mouse leukemias can be easily characterized as being of T origin by typing for Thy-I marker [18,19]. In this report we have confirmed that Thy-! antibodies discriminate between thymic and non-thymic lymphomas. However, Thy-I epitopes have been detected in a significant proportion of reticulum cell sarcomas and myeloid leukemias. Likewise, Walker et al. [20] recently demonstrated that a long-transplanted WEH1-3B cell line originally isolated from a myelomonocytic leukemia was Thy-l-positive. Ponzio et al. [21] communicated that some spontaneously occurring reticulosarcomas of the SJL strain have been typed as Thy-1 , whereas Robert and D u m o n t [22] observed that Thy-I + cells were frequently present in reticulum sarcoma cells of SJ L mice. In the light of some recent findings, our results demonstrating the presence of Thy-l.2 epitopes on some myeloid leukemias and reticulum cell sarcomas are not surprising. Both reticulum cell sarcomas and myeloid leukemias originate in the bone marrow. A significant number (25-30%) of mouse bone marrow cells including multipotential stem cells, hemopoietic progenitor cells and immature cells of the granulocyte/macrophage lineage has been reported as Thy1+ [9,10]. A number of growth factor-dependent lines of myeloid cells generated from long-term mouse bone marrow cultures were also found to be Thy-l-positive [23]. Similar observations have been made in the rat [24 28]. Hence, we can assume that malignant conversion of Thy-1 + precursor cells can give rise to the Thy-I + reticulum cell sarcomas and

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