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The binding of Maclura pomifera lectin to cells of the T-lymphocyte lineage in the rat
CELLULAR
IMMUNOLOGY
67, 101-l 11 (1982)
The Binding of Maclura pomifera Lectin to Cells of the T-Lymphocyte Lineage in the Rat’ RANDALL
W. BARTON
Department of Medicine, Pulmonary Division, University of Connecticut Health Center, Farmington, Connecticut 06032 Received August 25, 1981; accepted December 6, 1981 The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymph node were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT’ bone marrow cells, putative thymocyte progenitors, were MP+. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.
INTRODUCTION In rat lymphoid cells the predominant cell surface glycoproteins which are heavily glycosylated have been identified as differentiation antigens (1). Such differentiation antigens can be labeled by antibodies (2-4) or, being heavily glycosylated, lectins can be used as a means of identifying alterations in the cell surface during differentiation. The Maclura pomifera lectin (MP),’ isolated from seeds of the osage orange, has been reported to bind preferentially to T cells in the rat (5). The lectin has been reported to bind to the galactose-containing oligosaccharides, melibiose and stachyose, as well as a number of derivatives of galactose (5). Thymus cells bound larger quantities of MP lectin than cells of lymph node, spleen, or bone marrow, and the binding by spleen cells from thymectomized rats was reduced. However, no direct comparison has been made between the MP lectin and other known rat ’ This work was supported by a grant from the American Cancer Society (CHl 1 IA). ’ Abbreviations used: MP, Maclura pomifera lectin; TdT, terminal deoxynucleotidyl transferase; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; FAG, fluorescenceactivated cell sorter. 101 OOOS-8749/82/030101-I
1$02.00/O
Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
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T-cell and B-cell reagents. Nor is the distribution of MP binding to cells at various differentiation stages within the T-lymphocyte developmental lineage known. In the present study the tissue, anatomic, and developmental distribution of MP lectin binding is presented. The MP lectin binds to T lymphocytes and not B lymphocytes and is present on prethymic, thymic, as well as post-thymic cells of the T-lymphocyte lineage. The lectin also appears to bind, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/ 13 monoclonai antibody (3). MATERIALS
AND
METHODS
Animals and cells. Six- to eight-week-old Lewis and (Lewis X DA) F, hybrid rats of both sexes were used in all experiments. Gross virus-induced thymic lymphomas were obtained as previously described (6). Cell suspensions were prepared from thymus, spleen, and bone marrow as previously described (7), and washed in a balanced salt solution (BBSS) consisting of the following: 0.145 M NaCl, 2.7 mM KCI, 5.6 mM glucose, 0.36 mM NaH2P04, 0.9 mM CaC&, 0.5 rnM MgC12, 5 mM Tris-HCl, pH 7.2. Lectins and antibodies. Maclura pomifera lectin conjugated with fluorescein isothiocyanate (FITC) was obtained from Pierce Chemical Company (Rockford, Ill.). The mouse monoclonal antibodies W3/13, W3/25, and OX 8 which react with rat T cells and their subsets (2, 3) were obtained from Accurate Scientific (Hicksville, N.Y.). An F(ab’)l fraction of rabbit antiserum to rat brain Thy-l was prepared as previously described (8). An IgG fraction of rabbit antiserum to calf thymus terminal deoxynucleotidyl transferase (TdT) was kindly provided by Dr. F. J. Bollum. Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated and FITC-conjugated F(ab’)z fraction of goat anti-rat IgG (heavy and light chains), TRITC- and FITC-conjugated F(ab’), fractions of goat anti-rabbit IgG (heavy and light chains) and TRITCand FITC-conjugated F(ab’)z fractions of goat antimouse IgG (heavy and light chains), were purchased from Cappel Laboratories, Downingtown, Pennsylvania. TRITC-conjugated MP lectin was prepared by dissolving 0.5 mg of MP lectin (Pierce Chemical) in 1 ml of 0.15 M sodium phosphate, pH 9.5, adding 5 ~1 of a TRITC (Research Organics, Cleveland, Ohio) solution (30 mg/ml of DMSO) and incubation for 1 hr at room temperature in the dark. The pH was monitored at 0 and 30 min of incubation and the pH was adjusted to 9.5, if necessary, by the addition of 0.15 M Na,P04. After the incubation the lectin solution was dialyzed for 36 hr against a solution containing 0.01 M potassium phosphate, pH 7.5, and 0.05 M NaCl. The titer of the TRITC-MP lectin was virtually identical with that of the commercial FITC-MP lectin. Immunofluorescent staining of cell suspensions. Direct and indirect single-label immunofluorescence was performed by incubating 1 to 2 X lo6 cells for 20 min at 4°C with 10 ~1 of the appropriate antibody or lectin. The cells were then washed twice with BBSS containing 0.02 M NaN,. For indirect immunofluorescence the cells were then incubated with 10 ~1 of the appropriate fluorescent antibody and then washed twice with the BBSS + 0.02 M NaN3. To detect two antigenic specificities on cells the above step(s) were performed as described above followed by incubation with the second specific antibody or lectin, washed twice and, for indirect fluorescence, by incubation with a fluorescent-
Maclura
pomifera
LECTIN
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TO RAT T CELLS
103
conjugated antibody and washing. For example, in order to determine whether the MP lectin and W3/ 13 antibody stained overlapping populations of cells, cells were incubated with 10 ~1 of FITC-MP, washed twice, and incubated with 10 ~1 of W3/ 13. Following washing the cells were incubated with either FITC-F(ab’)2 goat antimouse IgG for analysis with fluorescence-activated cell sorter (FACS) or TRITCF( ab’), goat anti-mouse IgG for dual-color immunofluorescence microscopy. Analysis of cells was performed by the fluorescence-activated cell sorter (FACS IV, Becton-Dickinson, Sunnyvale, Calif.) and by fluorescence microscopy. FACS settings were Laser power 200 mW, Scatter gain 4/O.& fluorescence gain 16/0.5. Fluorescence microscopy was performed with a Zeiss RA microscope equipped for uv epi-illumination with a 50-W mercury vapor lamp. Zmmunojluorescence of frozen sections. Frozen sections of thymus, spleen, and lymph node were incubated with FITC-MP (3.0 hg/ml) for 15 min and washed for 3 min twice with BBSS. Controls consisted of sections similarly treated with FITC-MP which had been preincubated with melibiose, 5 mg/ml. Zmmunojluorescence of cell surface antigens and terminal deoxynucleotidyl transferase (TdT). The binding of the MP lectin and W3/13 antibody to TdT-
containing bone marrow cells was determined by dual immunofluorescence. Cell suspensions were first labeled with either TRIIC-MP or W3/13 antibody plus TRITC-F(ab’)* goat anti-mouse IgG (H and L chains) as described above. Cytocentrifuge smears of these stained cells were then made (Cytospin, Shandon, Sewickley, Pa.), fixed, and stained for TdT as described previously (9) using an IgG fraction of rabbit anti-TdT plus FITC-F(ab’), goat anti-rabbit IgG (H and L chains). MP lectin affinity chromatography. A suspension of 5.0 X lo8 thymocytes was suspended in 25 ~11of 0.1% Lubrol PX in 10 mM Tris-HCl, pH 8.0, to solubilize the cell glycoproteins (1). The suspension was then centrifuged at 3000g for 30 min and the supernatant was then centrifuged at 125,000g for 30 min. A MP lectin affinity column was prepared using activated Sepharose 4B (Pharmacia). A column of 0.4 ml of beads containing approximately 1 mg of MP lectin was used and 200 ~1 of detergent-solubilized material from 4.0 X lOa cells was added onto the column. The column was washed with 0.1% Lubrol PX in 10 mM Tris-HCl, pH 8.0, and eluted with 5 mg/ml melibiose in the same buffer. The starting material, the material not bound to the column, and the material eluted from the column were all tested for inhibition of W3/13 and anti-Thy1 antibodies. A 20-~1 volume of each sample was incubated with 20 ~1 of specific antibody (at a lo-fold higher concentration than used for immunofluorescence) for 4 hr at 4°C. The samples were then centrifuged at 12,000g for 30 min and the supernatants were diluted 5-fold and 10 ~1 was tested for binding to 2 X IO6 thymocytes by indirect immunofluorescence as described above. RESULTS Tissue distribution. The proportion of lymphoid cells binding the FITC-labeled MP lectin in various lymphoid tissues was examined both microscopically and with the fluorescence-activated cell sorter (Fig. 1). Virtually all thymocytes bind the lectin whereas lower proportions of lymph node cells and spleen cells are labeled. These percentages of positive cells did not change over a 50-fold concentration
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W. BARTON
FLUORESCENCE
INTENSITY
FIG. 1. Fluorescence histograms from FACS analysis of FITC-MP lectin binding to young adult rat lymphoid cell populations: (A) thymocytes, >98W; (B) lymph node cells, 65%; (C) spleen cells, 54%; (D) thymocytes incubated with FITC-MP + melibiose, 0.8%. The marker represents the separation of positive from negative.
range, 1.0 to 50 rg/ml of the fluorescent lectin. The specificity of binding was demonstrated by preincubation of the lectin with 5 mg/ml melibiose (disaccharide of galactopyranose and glucose) (Fig. 1) which reduced the percentage of positive cells to less than 1% in all tissues. Location of MP+ cells in spleen and lymph node. Frozen sections of spleen and lymph node were stained with the FITC-MP lectin to determine the anatomic location of MP-binding cells (Fig. 2). The ring fluorescence suggested surface membrane binding of the lectin. In cervical and mesenteric lymph nodes, the vast majority of cells throughout the paracortex were fluorescent whereas germinal center cells and follicular lymphocytes were negative. In the spleen, cells in the periarteriolar lymphatic sheath were positively stained. Conversely, follicular cells, germinal center cells, and marginal zone cells were negative. Within the follicles of both lymph node and spleen, large autofluorescent macrophages were seen. Their autofluorescence was yellow-orange in color, quite distinct from the apple green color of the FITC-MP-labeled cells. MP binding to thymus sections revealed a uniformly bright fluorescence throughout the entire section. No staining of these tissues was observed when the lectin was preincubated with melibiose. T and B lymphocyte distribution. Spleen and lymph node lymphocytes were singly and multiply labeled with the MP lectin, anti-rat immunoglobulin, and the monoclonal antibodies to rat T cells and their subsets, W3/13, W3/25, and OX 8 (2, 3), to determine the distribution of MP+ cells among T and B lymphocytes. The W3/13 antibody binds to all T cells and not B cells whereas W3/25 and OX 8 identify nonoverlapping T-cell subsets which together comprise all T cells (3). Cells which bind W3/25 have been found to exhibit helper cell activity, GVH
Macluru
pomifera LECTIN
BINDING
TO RAT T CELLS
FIG. 2. Frozen sections of spleen and lymph node exposed to FITC-MP lectin. (Top) Spleen section: lymphocytes in periarterolar lymphatic sheath (PALS) appear to exhibit membrane fluorescence whereas those in the follicle do not. (Bottom) Lymph node section: lymphocytes in the paracortex (PC) are stained whereas those in the follicle are not. The large bright spots are macrophages which autofluoresce yellowish orange.
activity, and mixed lymphocyte responsiveness (2), whereas OX 8 binding cells mediate allogeneic suppression (3) and have cytotoxic activity ( 10). Overlaps between MP+ cells and T and/or B cells were established by two methods: (i) comparing the percentage ofcells labeled by FITC-lectin and FITCantibodies singly with that obtained by multiple labeling using the fluorescenceactivated cell sorter; (ii) using dual-color immunofluorescence and determining the proportions of single- and dual-labeled cells by fluorescence microscopy. Using both
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W. BARTON
FLUORESCENCE
INTENSITY
FIG. 3. Fluorescence histograms from FACS analysis of the binding of MP lectin, anti-rat Ig and W3/13 monoclonal antibody to rat lymph node cells. (A) W3/13, 68.4%; (B) MP lectin, 68.9%; (C) W3/13 + MP, 69.6%; (D) anti-rat Ig, 27.4%; (E) anti-rat Ig + MP, 97.0%. The marker represents the separation of positive from negative.
of these techniques confirmed that the MP+ cells in spleen and lymph node were T cells and not B cells; all W?/ 13+, W3/25+, and OX 8+ cells in spleen and lymph node bound MP. For example, as shown in Fig. 3A-C, the percentage of lymph node cells which were positively stained by W3/13 (Fig. 3A) or by MP (Fig. 3B) alone was virtually identical, approximately 68-69%. When both reagents were incubated with an aliquot of cells the percentage of positive cells was essentially unchanged. However, the relative fluorescence intensity of the MP + W3/13-labeled cells was noticeably higher (Fig. 3C). This emphasizes the conclusion that both MP and W3/ 13 were binding to the same cells and, therefore, each positively stained cell was more fluorescent. When cells were labeled with both FITC-MP and FITC-anti-rat Ig, the percentage of positive cells equaled the sum of the percentages obtained when cells were singly labeled (Fig. 3B, D, and E). Thus, as shown in Fig. 3E, virtually 100% of lymph node cells were fluorescent when the cell suspension was multiply labeled with FITC-MP and FITC-anti-rat Ig. Dual-color immunofluorescence microscopy
A4uclura
pomifera
LECTIN
BINDING
TO RAT T CELLS
LYMPH
NODE
1
SPLEEN
L 0
20
40
PERCENTAGE
60 OF
107
a0
I 100
CELLS
4. Percentages of spleen and lymph node cells stained by MP lectin, anti-Thy1 and the monoclonal antibodies W3/13, W3/25, and OX 8. ‘a, MP; 111, W3/13; I, Thyl; B, OX 8; a, W3/25. MP and W3/13 stain the same cell population, virtually all, if not all, T cells. A minority of such T cells are Thyl+ and Thy1 is present on a proportion of both the W3/25+ and OX 8+ T-cell subsets. FIG.
confirmed that no small population existed.
of MP+, Igf, lymph node, or spleen lymphocytes
T lymphocyte subpopulations. The Thy1 antigen has been reported to be present on a minority of peripheral T cells in the rat (4). The proportion of MP+, W3/ 13+, W3/25+, and OX 8+ cells in lymph node and spleen which were Thyl+ was determined (Fig. 4). Almost identical proportions of MP+ cells and W3/13+ cells were Thyl+, 16- 17% in spleen and 13-14s in lymph node. Although Thy1 + cells were not restricted exclusively to either the W3/25+ or the OX 8+ T-cell subset, a higher proportion of OX 8+ cells were Thyl+. In spleen approximately 24% of OX 8+ cells were Thyl+ as compared to 16% of W3/25+ cells. Similarly, in lymph node 23% of OX 8+ cells were Thyl+ and 12% of W3/25+ cells were Thyl+. TdF thymocyte progenitors in bone marrow. In normal animals, bone marrow cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) are believed to be thymocyte progenitors. The proportion of TdT+ cells in bone marrow which were MP+ or W3/13+ was determined by dual-color immunofluorescence microscopy. Bone marrow cells were stained in suspension with W3/13 or MP labeled with TRITC; cytocentrifuge smears were then made, fixed, and stained for TdT labeled with FITC. Essentially identical proportions of TdT+ cells were found to be MP+ and W3/13+ (Table 1). Also, the MPf, TdT+ cells and W3/ 13+, TdT+ cells appeared to be the same cells since staining cell suspensions with both W3/ 13 and MP yielded the same proportion as when cells were singly labeled with either MP or W3/13 (Table 1). No TdT+ cells were found to be W3/25+ or OX 8+. Gross virus-induced rat thymic lymphomas were also tested for their binding of MP lectin; in a previous study these lymphoma leukemias were shown to have a prethymic origin (6). Like normal thymocytes, these TdT+ neoplastic thymocytes were MP+. In all nine lymphomas tested greater than 95% of the cells were stained. Coincident binding of W3/13 and MP. Since all experiments had shown coincident binding of MP lectin and W3/ 13 antibody, and since W3/ 13 has been shown
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TABLE
1
Surface Markers on TdT+ Bone Marrow Cells Treatment
Proportion (%) of TdT+ cells stained
MP w3/13 MP + W3/13
23.5 (20.2-31.4) 22.0 (19-33.3) 23.2 (19532.2)
’ Percentage of TdT+ cells which were labeled by either the MP lectin or W3/13 antibody; the minimum and maximum values found are in parentheses. Surface markers were labeled with TRITC and TdT was detected with FITC; see Materials and Methods for details.
to bind to both T cells and members of the granuloid series in bone marrow (1 l), the binding of both MP and W3/13 to bone marrow was examined (Table 2). In bone marrow as in the other tissues examined, MP and W3/13 stained the same cells since the percentage of cells stained was not increased when the W3/ 13 and MP were added together as compared to the percentages stained by each separately. The possibility that the coincident binding of W3/13 and MP to cells was a result of binding to the same cell surface glycoproteins was examined. Solubilized thymocyte glycoprotein preparations were passed through an MP lectin affinity column and the bound material was eluted with melibiose. An aliquot of the initial unpassed preparation, the material not bound to the column, and the material eluted from the column were tested for their ability to inhibit the binding of W3/ 13 and anti-Thy1 antibodies to thymocytes as assessed by immunofluorescence (Table 3). Anti-Thy1 antibodies were tested because the carbohydrate composition of the Thy1 glycoprotein is distinctly different from that of the W3/13 molecule and Thy1 would not be predicted to bind to the MP lectin. Both W3/13 and antiThy1 were inhibited by the initial unpassed membrane preparation. However, the material which did not bind to the MP column inhibited only anti-Thy1 but not W3/ 13. In contrast, the material which was bound and eluted from the MP column inhibited only the W3/13 and not the anti-Thy1 antibodies. DISCUSSION The results of the present study clearly demonstrate a nonimmunoglobulin agent, lectin isolated from A4ucluru pomifera seeds, which binds to rat T cells not B cells in spleen and lymph node. A previous study by Jones and Feldman showed that thymocytes bound the MP lectin to the greatest extent followed TABLE
2
Binding of MP and W3/13 to Rat Bone Marrow Cells Treatment
Percentage cells stained’
MP w3/13 MP + W3/13
39 (30-46) 37 (31-44) 39 (39-45)
a Represents mean value with minimum and maximum values in parentheses.
rebut (5) by
Muclura
pomifera
LECTIN
BINDING
TABLE
109
TO RAT T CELLS
3
Analysis of Thymocyte Glycoproteins Binding to MP Lectin Inhibition of antibody binding to thymocytes” Fraction
Anti-Thy 1
w3/13
Total solubilized extract Fraction unbound to MP-Sepharose Fraction bound and eluted from MP-Sepharose
+ + -
+ +
’ The various fractions were tested for their ability to inhibit anti-Thy1 and W3/ 13 antibody binding to thymocytes. (-) >75% of cells stained; (+) ~25% of cells stained. See Materials and Methods for details.
lymph node, spleen, and bone marrow, respectively. The contention that the MPbinding cells in peripheral lymphoid tissue were T cells was supported by the observation that spleens from neonatally thymectomized rats had reduced numbers of MP-binding cells. In the present study the selective binding to peripheral T cells but not B cells was shown by direct comparison with antibodies specific for rat T cells and B cells. In spleen and lymph node MP+ cells and sIg+ cells represent distinct, nonoverlapping populations. This was shown both by single-color additive experiments with the FACS and by dual-color immunofluorescence microscopy. In contrast, all lymph node and spleen cells binding the W3/13 monoclonal antibody, which reacts with most, if not all, peripheral T cells (3), were MP+. Similarly, all W3/25+ and OX 8+ lymph node and spleen cells, which represent nonoverlapping peripheral T-cell subsets (2, 3), were MP+. Thus, all peripheral T cells, as identified by these monoclonal antibodies, bound the MP lectin. The anatomic distribution of MP in spleen and lymph node also supported the T-cell specificity. The thymus-dependent areas, the paracortex in lymph node and periarteriolar lymphatic sheath in spleen (12), were stained by the lectin, whereas the B-cell areas, the lymphoid follicles and germinal centers (12), in both tissues were not. Cells at differing stages within the T-cell developmental lineage were also MP+. Virtually all thymocytes bound the MP lectin which corroborates the findings of Jones and Feldman (5). Also, no differences in fluorescence intensity were seen between cortex and medulla of frozen thymus sections. Interestingly, approximately 20-25s of bone marrow cells containing terminal deoxynucleotidyl transferase (TdT) were MP+ and W3/13+. TdT is restricted to cortical thymocytes (13) and to a small subset of “null” cells in bone marrow and prepubertal spleen (14). Inasmuch as TdT+ bone marrow cells are present in normal proportions in congenitally athymic mice and neonatally thymectomized rats (14) and they can be induced to express thymus-specific cell surface antigens by thymic humoral factors (15) bone marrow TdT+ cells are believed to be thymocyte progenitors. The significance of a subset of TdT+ bone marrow cells being MPf, W3/ 13+ remains to be established. It seems unlikely that these are thymus-derived cells circulating from thymus to bone marrow (16) since they are W3/25-, OX 8- and all thymocytes as well as peripheral T cells are W3/25+ and/or OX 8+ (3). It is more tempting to speculate that the MP+, TdT+ bone marrow cells represent the most
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mature population of prothymocytes immediately prior to their emigration from bone marrow to thymus. This is reminiscent of thymosin induction studies by Goldschneider et al. in the mouse, in which “early” and “late” TdT+ prothymocytes were postulated (15). Both the late TdT+ cells in mouse bone marrow and the MP+, W3/ 13+ TdT+ rat bone marrow cells may serve as the immediate precursors of TdT+ thymocytes. It has been shown previously that thymic-dependent Gross virus (RAGV)-induced lymphoma leukemias in the rat may have a prethymic origin (6). These TdT+ neoplastic thymocytes contained a subband of the LDH-5 isozyme (LDH5’) that was not detected in normal thymocytes but which was present in fractions of bone marrow and prepubertal spleen cells enriched for TdT+ cells. Since many of the leukemic cells have the same phenotype as the minor population of TdT+ bone marrow cells described in the present study, it will be clearly of interest to determine if the MP+, W3/13+ TdTf bone marrow cells contain the LDH-5’. If so, it is possible that such cells are the targets of leukemic transformation by the RAGV. In a previous study, Thy1 was demonstrated on a minority of peripheral T cells as defined by the heteroantiserum ALSr (4). In the present study, similar proportions of Thyl+ T cells were found using both the W3/13 antibody and the MP lectin to identify T cells. In both studies there was a higher proportion of Thyl+ T cells in spleen than in lymph node. The Thy+ T cells were not found exclusively in either the W3/25+ or OX 8+ T-cell subset. However, a higher proportion of OX 8+ cells were Thyl+ than were W3/25+ cells. Thus, four phenotypic subsets of rat peripheral T cells can be described: (1) MP+, W3/25+, OX 8-, Thyl-; (2) MP+, W3/25-, OX 8+, Thyl-; (3) MP+, W3/25+, OX 8-, Thyl’; (4) MP+, W3/ 25-, OX 8+, Thyl’. Cells which bind W3/25 antibody exhibit helper cell activity, GVH activity, and mixed lymphocyte responsiveness (2, 3), whereas OX 8 binding cells mediate allogeneic suppression (2, 3), and have cytotoxic activity (10). Whether the four phenotypic subsets of rat peripheral T cells described above represent functionally distinct populations or whether the Thyl+ T cells are simply more recently derived from the thymus remains to be established. However, Thyl+ T cells in the rat have been reported to exhibit suppressor cell activity (17). Thus, it is tempting to speculate that functional T-cell subpopulations may be described by these four cell surface phenotypes. The specificity of binding of the MP lectin has been described previously (18). The oligosaccharides, melibiose (a disaccharide of D-galactopyranose and D-ghcase) and stachyose (a tetrasaccharide of 2-D-galactose residues, D-glucose and Dfructose), and the monosaccharide N-acetyl-galactosamine were the most potent inhibitors found. All derivatives of D-galactose exhibited significant inhibition of MP binding whereas glucose and its derivatives as well as a number of other monoand oligosaccharides were not inhibitory. The chemical composition of the W3/ 13 antigen has been described recently (19). Carbohydrate comprises 60% of the molecule and consists almost exclusively of galactose, galactosamine, and sialic acid. This carbohydrate composition is distinct from that of other known cell surface glycoproteins such as the Thy1 and leukocyte-common glycoproteins ( 19). Given the distinct carbohydrate composition of the W3/ 13 antigen and the binding specificity of the MP lectin, it is not surprising that the thymocyte glycoproteins
Maclura
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which were isolated by MP lectin affinity chromatography are able to inhibit the W3/13 antibody but not the anti-Thy1 antibody. Antibodies to the Thy1 antigen were run as a control since the Thy1 carbohydrate composition is distinct from that of the W3/ 13 glycoprotein and Thy1 would be less likely to bind to the MP column. It might be argued that the repeated washing of the MP column during the elution process resulted in the nonspecific “leakage” of lectin or lectin-glycoprotein complexes. This possibility was tested by substituting glucose for melibiose during the elution process. The resulting “eluate” did not inhibit either W3/13 or anti-Thy1 antibodies. An additional control, which was not done for logistical reasons, would have been to pass a solubilized B-cell glycoprotein preparation through a MP lectin column and to test the unbound and the melibiose-eluted fractions for their ability to inhibit W3/13. Nevertheless, the present results indicate that the W3/13 glycoprotein binds to the MP lectin. However, it is not known whether the MP lectin is binding exclusively to the W3/13 glycoprotein or simply that the W3/13 glycoprotein is one of a number of glycoproteins which binds to MP. Studies are in progress to identify the cell surface molecule(s) which are bound by the MP lectin. ACKNOWLEDGMENTS The author gratefully acknowledges the excellent technical assistance of Maria Gionfriddo and the excellent assistance of Mrs. Barbara Hoekman in the preparation of the manuscript.
REFERENCES 1. Standring, R., McMaster, W. R., Sunderland, C. A., and Williams, A. F., Eur. J. Zmmunol. 8, 832, 1978. 2. Brideau, R. J., Carter, P. B., McMaster, W. R., Mason, D. W., and Williams, A. F., Eur. .I. Immunol. 10, 609, 1980. 3. Mason, D. W., Brideau, R. J., McMaster, W. R., Webb, M., White, R. A. H., and Williams, A. F., In “Monoclonal Antibodies” (R. H. Kennett and T. J. McKearn, Eds.), pp. 251-273. Plenum, New York, 1980. 4. Ritter, M. A., Gordon, L. K., and Goldschneider, I., J. Immunol. 121, 2463, 1978. 5. Jones, J. M., and Feldman, J. D., J. Immunol. 111, 1765, 1973. 6. Barton, R. W., Tausche, F., and Goldschneider, I., J. Immune!. 125, 2299, 1980. 7. Barton, R. W., Martinuik, F., Hirschhorn, R., and Goldschneider, I., J. Immunol. 122, 216, 1979. 8. Goldschneider, I., Gregoire, K. E., Barton, R. W., and Bollum, F. J., Proc. Nat. Acad. Sci. USA 74, 734, 1977. 9. Gregoire, K. E., Goldschneider, I., Barton, R. W., and Bollum, F. J., Proc. Nat. Acad. Sci. USA 74, 3993, 1977. 10. Woan, M., McGregor, D. D., and Goldschneider, I., J. Immunol. 127, 2330, 1981. il. Williams, A. F., Galfre, G., and Milstein, C., Cell 12, 663, 1977. 12. Goldschneider, I., and McGregor, D. D., J. Exp. Med. 138, 1443, 1973. 13. Barton, R. W., Goldschneider, I., and Bollum, F. J., J. Immunol. 116, 462, 1976. 14. Gregoire, K. E., Goldschneider, I., Barton, R. W., and Bollum, F. J., J. Immunol. 123, 1347, 1979. 15. Goldschneider, I., Ahmed, A., Bollum, F. J., and Goldstein, A. L., Proc. Nut. Acud. Sci. USA 78, 2469, 1981. 16. Order, S. E., and Waksman, B. H., Transplantation 8, 783, 1969. 17. Goldschneider, I., and Bollum, F. J., In “4th International Congress of Immunology (Abstracts), Paris, 1980.” 18. Cawley, L. P., Jones, J. M., and Teresa, G. W., Transfusion 7, 343, 1967. 19. Brown, W. R. A., Barclay, A. N., Sunderland, C. A., and Williams, A. F., Nature (London) 289, 456, 1981.
IMMUNOLOGY
67, 101-l 11 (1982)
The Binding of Maclura pomifera Lectin to Cells of the T-Lymphocyte Lineage in the Rat’ RANDALL
W. BARTON
Department of Medicine, Pulmonary Division, University of Connecticut Health Center, Farmington, Connecticut 06032 Received August 25, 1981; accepted December 6, 1981 The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymph node were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT’ bone marrow cells, putative thymocyte progenitors, were MP+. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.
INTRODUCTION In rat lymphoid cells the predominant cell surface glycoproteins which are heavily glycosylated have been identified as differentiation antigens (1). Such differentiation antigens can be labeled by antibodies (2-4) or, being heavily glycosylated, lectins can be used as a means of identifying alterations in the cell surface during differentiation. The Maclura pomifera lectin (MP),’ isolated from seeds of the osage orange, has been reported to bind preferentially to T cells in the rat (5). The lectin has been reported to bind to the galactose-containing oligosaccharides, melibiose and stachyose, as well as a number of derivatives of galactose (5). Thymus cells bound larger quantities of MP lectin than cells of lymph node, spleen, or bone marrow, and the binding by spleen cells from thymectomized rats was reduced. However, no direct comparison has been made between the MP lectin and other known rat ’ This work was supported by a grant from the American Cancer Society (CHl 1 IA). ’ Abbreviations used: MP, Maclura pomifera lectin; TdT, terminal deoxynucleotidyl transferase; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; FAG, fluorescenceactivated cell sorter. 101 OOOS-8749/82/030101-I
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Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
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T-cell and B-cell reagents. Nor is the distribution of MP binding to cells at various differentiation stages within the T-lymphocyte developmental lineage known. In the present study the tissue, anatomic, and developmental distribution of MP lectin binding is presented. The MP lectin binds to T lymphocytes and not B lymphocytes and is present on prethymic, thymic, as well as post-thymic cells of the T-lymphocyte lineage. The lectin also appears to bind, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/ 13 monoclonai antibody (3). MATERIALS
AND
METHODS
Animals and cells. Six- to eight-week-old Lewis and (Lewis X DA) F, hybrid rats of both sexes were used in all experiments. Gross virus-induced thymic lymphomas were obtained as previously described (6). Cell suspensions were prepared from thymus, spleen, and bone marrow as previously described (7), and washed in a balanced salt solution (BBSS) consisting of the following: 0.145 M NaCl, 2.7 mM KCI, 5.6 mM glucose, 0.36 mM NaH2P04, 0.9 mM CaC&, 0.5 rnM MgC12, 5 mM Tris-HCl, pH 7.2. Lectins and antibodies. Maclura pomifera lectin conjugated with fluorescein isothiocyanate (FITC) was obtained from Pierce Chemical Company (Rockford, Ill.). The mouse monoclonal antibodies W3/13, W3/25, and OX 8 which react with rat T cells and their subsets (2, 3) were obtained from Accurate Scientific (Hicksville, N.Y.). An F(ab’)l fraction of rabbit antiserum to rat brain Thy-l was prepared as previously described (8). An IgG fraction of rabbit antiserum to calf thymus terminal deoxynucleotidyl transferase (TdT) was kindly provided by Dr. F. J. Bollum. Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated and FITC-conjugated F(ab’)z fraction of goat anti-rat IgG (heavy and light chains), TRITC- and FITC-conjugated F(ab’), fractions of goat anti-rabbit IgG (heavy and light chains) and TRITCand FITC-conjugated F(ab’)z fractions of goat antimouse IgG (heavy and light chains), were purchased from Cappel Laboratories, Downingtown, Pennsylvania. TRITC-conjugated MP lectin was prepared by dissolving 0.5 mg of MP lectin (Pierce Chemical) in 1 ml of 0.15 M sodium phosphate, pH 9.5, adding 5 ~1 of a TRITC (Research Organics, Cleveland, Ohio) solution (30 mg/ml of DMSO) and incubation for 1 hr at room temperature in the dark. The pH was monitored at 0 and 30 min of incubation and the pH was adjusted to 9.5, if necessary, by the addition of 0.15 M Na,P04. After the incubation the lectin solution was dialyzed for 36 hr against a solution containing 0.01 M potassium phosphate, pH 7.5, and 0.05 M NaCl. The titer of the TRITC-MP lectin was virtually identical with that of the commercial FITC-MP lectin. Immunofluorescent staining of cell suspensions. Direct and indirect single-label immunofluorescence was performed by incubating 1 to 2 X lo6 cells for 20 min at 4°C with 10 ~1 of the appropriate antibody or lectin. The cells were then washed twice with BBSS containing 0.02 M NaN,. For indirect immunofluorescence the cells were then incubated with 10 ~1 of the appropriate fluorescent antibody and then washed twice with the BBSS + 0.02 M NaN3. To detect two antigenic specificities on cells the above step(s) were performed as described above followed by incubation with the second specific antibody or lectin, washed twice and, for indirect fluorescence, by incubation with a fluorescent-
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conjugated antibody and washing. For example, in order to determine whether the MP lectin and W3/ 13 antibody stained overlapping populations of cells, cells were incubated with 10 ~1 of FITC-MP, washed twice, and incubated with 10 ~1 of W3/ 13. Following washing the cells were incubated with either FITC-F(ab’)2 goat antimouse IgG for analysis with fluorescence-activated cell sorter (FACS) or TRITCF( ab’), goat anti-mouse IgG for dual-color immunofluorescence microscopy. Analysis of cells was performed by the fluorescence-activated cell sorter (FACS IV, Becton-Dickinson, Sunnyvale, Calif.) and by fluorescence microscopy. FACS settings were Laser power 200 mW, Scatter gain 4/O.& fluorescence gain 16/0.5. Fluorescence microscopy was performed with a Zeiss RA microscope equipped for uv epi-illumination with a 50-W mercury vapor lamp. Zmmunojluorescence of frozen sections. Frozen sections of thymus, spleen, and lymph node were incubated with FITC-MP (3.0 hg/ml) for 15 min and washed for 3 min twice with BBSS. Controls consisted of sections similarly treated with FITC-MP which had been preincubated with melibiose, 5 mg/ml. Zmmunojluorescence of cell surface antigens and terminal deoxynucleotidyl transferase (TdT). The binding of the MP lectin and W3/13 antibody to TdT-
containing bone marrow cells was determined by dual immunofluorescence. Cell suspensions were first labeled with either TRIIC-MP or W3/13 antibody plus TRITC-F(ab’)* goat anti-mouse IgG (H and L chains) as described above. Cytocentrifuge smears of these stained cells were then made (Cytospin, Shandon, Sewickley, Pa.), fixed, and stained for TdT as described previously (9) using an IgG fraction of rabbit anti-TdT plus FITC-F(ab’), goat anti-rabbit IgG (H and L chains). MP lectin affinity chromatography. A suspension of 5.0 X lo8 thymocytes was suspended in 25 ~11of 0.1% Lubrol PX in 10 mM Tris-HCl, pH 8.0, to solubilize the cell glycoproteins (1). The suspension was then centrifuged at 3000g for 30 min and the supernatant was then centrifuged at 125,000g for 30 min. A MP lectin affinity column was prepared using activated Sepharose 4B (Pharmacia). A column of 0.4 ml of beads containing approximately 1 mg of MP lectin was used and 200 ~1 of detergent-solubilized material from 4.0 X lOa cells was added onto the column. The column was washed with 0.1% Lubrol PX in 10 mM Tris-HCl, pH 8.0, and eluted with 5 mg/ml melibiose in the same buffer. The starting material, the material not bound to the column, and the material eluted from the column were all tested for inhibition of W3/13 and anti-Thy1 antibodies. A 20-~1 volume of each sample was incubated with 20 ~1 of specific antibody (at a lo-fold higher concentration than used for immunofluorescence) for 4 hr at 4°C. The samples were then centrifuged at 12,000g for 30 min and the supernatants were diluted 5-fold and 10 ~1 was tested for binding to 2 X IO6 thymocytes by indirect immunofluorescence as described above. RESULTS Tissue distribution. The proportion of lymphoid cells binding the FITC-labeled MP lectin in various lymphoid tissues was examined both microscopically and with the fluorescence-activated cell sorter (Fig. 1). Virtually all thymocytes bind the lectin whereas lower proportions of lymph node cells and spleen cells are labeled. These percentages of positive cells did not change over a 50-fold concentration
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FIG. 1. Fluorescence histograms from FACS analysis of FITC-MP lectin binding to young adult rat lymphoid cell populations: (A) thymocytes, >98W; (B) lymph node cells, 65%; (C) spleen cells, 54%; (D) thymocytes incubated with FITC-MP + melibiose, 0.8%. The marker represents the separation of positive from negative.
range, 1.0 to 50 rg/ml of the fluorescent lectin. The specificity of binding was demonstrated by preincubation of the lectin with 5 mg/ml melibiose (disaccharide of galactopyranose and glucose) (Fig. 1) which reduced the percentage of positive cells to less than 1% in all tissues. Location of MP+ cells in spleen and lymph node. Frozen sections of spleen and lymph node were stained with the FITC-MP lectin to determine the anatomic location of MP-binding cells (Fig. 2). The ring fluorescence suggested surface membrane binding of the lectin. In cervical and mesenteric lymph nodes, the vast majority of cells throughout the paracortex were fluorescent whereas germinal center cells and follicular lymphocytes were negative. In the spleen, cells in the periarteriolar lymphatic sheath were positively stained. Conversely, follicular cells, germinal center cells, and marginal zone cells were negative. Within the follicles of both lymph node and spleen, large autofluorescent macrophages were seen. Their autofluorescence was yellow-orange in color, quite distinct from the apple green color of the FITC-MP-labeled cells. MP binding to thymus sections revealed a uniformly bright fluorescence throughout the entire section. No staining of these tissues was observed when the lectin was preincubated with melibiose. T and B lymphocyte distribution. Spleen and lymph node lymphocytes were singly and multiply labeled with the MP lectin, anti-rat immunoglobulin, and the monoclonal antibodies to rat T cells and their subsets, W3/13, W3/25, and OX 8 (2, 3), to determine the distribution of MP+ cells among T and B lymphocytes. The W3/13 antibody binds to all T cells and not B cells whereas W3/25 and OX 8 identify nonoverlapping T-cell subsets which together comprise all T cells (3). Cells which bind W3/25 have been found to exhibit helper cell activity, GVH
Macluru
pomifera LECTIN
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TO RAT T CELLS
FIG. 2. Frozen sections of spleen and lymph node exposed to FITC-MP lectin. (Top) Spleen section: lymphocytes in periarterolar lymphatic sheath (PALS) appear to exhibit membrane fluorescence whereas those in the follicle do not. (Bottom) Lymph node section: lymphocytes in the paracortex (PC) are stained whereas those in the follicle are not. The large bright spots are macrophages which autofluoresce yellowish orange.
activity, and mixed lymphocyte responsiveness (2), whereas OX 8 binding cells mediate allogeneic suppression (3) and have cytotoxic activity ( 10). Overlaps between MP+ cells and T and/or B cells were established by two methods: (i) comparing the percentage ofcells labeled by FITC-lectin and FITCantibodies singly with that obtained by multiple labeling using the fluorescenceactivated cell sorter; (ii) using dual-color immunofluorescence and determining the proportions of single- and dual-labeled cells by fluorescence microscopy. Using both
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FIG. 3. Fluorescence histograms from FACS analysis of the binding of MP lectin, anti-rat Ig and W3/13 monoclonal antibody to rat lymph node cells. (A) W3/13, 68.4%; (B) MP lectin, 68.9%; (C) W3/13 + MP, 69.6%; (D) anti-rat Ig, 27.4%; (E) anti-rat Ig + MP, 97.0%. The marker represents the separation of positive from negative.
of these techniques confirmed that the MP+ cells in spleen and lymph node were T cells and not B cells; all W?/ 13+, W3/25+, and OX 8+ cells in spleen and lymph node bound MP. For example, as shown in Fig. 3A-C, the percentage of lymph node cells which were positively stained by W3/13 (Fig. 3A) or by MP (Fig. 3B) alone was virtually identical, approximately 68-69%. When both reagents were incubated with an aliquot of cells the percentage of positive cells was essentially unchanged. However, the relative fluorescence intensity of the MP + W3/13-labeled cells was noticeably higher (Fig. 3C). This emphasizes the conclusion that both MP and W3/ 13 were binding to the same cells and, therefore, each positively stained cell was more fluorescent. When cells were labeled with both FITC-MP and FITC-anti-rat Ig, the percentage of positive cells equaled the sum of the percentages obtained when cells were singly labeled (Fig. 3B, D, and E). Thus, as shown in Fig. 3E, virtually 100% of lymph node cells were fluorescent when the cell suspension was multiply labeled with FITC-MP and FITC-anti-rat Ig. Dual-color immunofluorescence microscopy
A4uclura
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NODE
1
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L 0
20
40
PERCENTAGE
60 OF
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I 100
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4. Percentages of spleen and lymph node cells stained by MP lectin, anti-Thy1 and the monoclonal antibodies W3/13, W3/25, and OX 8. ‘a, MP; 111, W3/13; I, Thyl; B, OX 8; a, W3/25. MP and W3/13 stain the same cell population, virtually all, if not all, T cells. A minority of such T cells are Thyl+ and Thy1 is present on a proportion of both the W3/25+ and OX 8+ T-cell subsets. FIG.
confirmed that no small population existed.
of MP+, Igf, lymph node, or spleen lymphocytes
T lymphocyte subpopulations. The Thy1 antigen has been reported to be present on a minority of peripheral T cells in the rat (4). The proportion of MP+, W3/ 13+, W3/25+, and OX 8+ cells in lymph node and spleen which were Thyl+ was determined (Fig. 4). Almost identical proportions of MP+ cells and W3/13+ cells were Thyl+, 16- 17% in spleen and 13-14s in lymph node. Although Thy1 + cells were not restricted exclusively to either the W3/25+ or the OX 8+ T-cell subset, a higher proportion of OX 8+ cells were Thyl+. In spleen approximately 24% of OX 8+ cells were Thyl+ as compared to 16% of W3/25+ cells. Similarly, in lymph node 23% of OX 8+ cells were Thyl+ and 12% of W3/25+ cells were Thyl+. TdF thymocyte progenitors in bone marrow. In normal animals, bone marrow cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) are believed to be thymocyte progenitors. The proportion of TdT+ cells in bone marrow which were MP+ or W3/13+ was determined by dual-color immunofluorescence microscopy. Bone marrow cells were stained in suspension with W3/13 or MP labeled with TRITC; cytocentrifuge smears were then made, fixed, and stained for TdT labeled with FITC. Essentially identical proportions of TdT+ cells were found to be MP+ and W3/13+ (Table 1). Also, the MPf, TdT+ cells and W3/ 13+, TdT+ cells appeared to be the same cells since staining cell suspensions with both W3/ 13 and MP yielded the same proportion as when cells were singly labeled with either MP or W3/13 (Table 1). No TdT+ cells were found to be W3/25+ or OX 8+. Gross virus-induced rat thymic lymphomas were also tested for their binding of MP lectin; in a previous study these lymphoma leukemias were shown to have a prethymic origin (6). Like normal thymocytes, these TdT+ neoplastic thymocytes were MP+. In all nine lymphomas tested greater than 95% of the cells were stained. Coincident binding of W3/13 and MP. Since all experiments had shown coincident binding of MP lectin and W3/ 13 antibody, and since W3/ 13 has been shown
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TABLE
1
Surface Markers on TdT+ Bone Marrow Cells Treatment
Proportion (%) of TdT+ cells stained
MP w3/13 MP + W3/13
23.5 (20.2-31.4) 22.0 (19-33.3) 23.2 (19532.2)
’ Percentage of TdT+ cells which were labeled by either the MP lectin or W3/13 antibody; the minimum and maximum values found are in parentheses. Surface markers were labeled with TRITC and TdT was detected with FITC; see Materials and Methods for details.
to bind to both T cells and members of the granuloid series in bone marrow (1 l), the binding of both MP and W3/13 to bone marrow was examined (Table 2). In bone marrow as in the other tissues examined, MP and W3/13 stained the same cells since the percentage of cells stained was not increased when the W3/ 13 and MP were added together as compared to the percentages stained by each separately. The possibility that the coincident binding of W3/13 and MP to cells was a result of binding to the same cell surface glycoproteins was examined. Solubilized thymocyte glycoprotein preparations were passed through an MP lectin affinity column and the bound material was eluted with melibiose. An aliquot of the initial unpassed preparation, the material not bound to the column, and the material eluted from the column were tested for their ability to inhibit the binding of W3/ 13 and anti-Thy1 antibodies to thymocytes as assessed by immunofluorescence (Table 3). Anti-Thy1 antibodies were tested because the carbohydrate composition of the Thy1 glycoprotein is distinctly different from that of the W3/13 molecule and Thy1 would not be predicted to bind to the MP lectin. Both W3/13 and antiThy1 were inhibited by the initial unpassed membrane preparation. However, the material which did not bind to the MP column inhibited only anti-Thy1 but not W3/ 13. In contrast, the material which was bound and eluted from the MP column inhibited only the W3/13 and not the anti-Thy1 antibodies. DISCUSSION The results of the present study clearly demonstrate a nonimmunoglobulin agent, lectin isolated from A4ucluru pomifera seeds, which binds to rat T cells not B cells in spleen and lymph node. A previous study by Jones and Feldman showed that thymocytes bound the MP lectin to the greatest extent followed TABLE
2
Binding of MP and W3/13 to Rat Bone Marrow Cells Treatment
Percentage cells stained’
MP w3/13 MP + W3/13
39 (30-46) 37 (31-44) 39 (39-45)
a Represents mean value with minimum and maximum values in parentheses.
rebut (5) by
Muclura
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LECTIN
BINDING
TABLE
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3
Analysis of Thymocyte Glycoproteins Binding to MP Lectin Inhibition of antibody binding to thymocytes” Fraction
Anti-Thy 1
w3/13
Total solubilized extract Fraction unbound to MP-Sepharose Fraction bound and eluted from MP-Sepharose
+ + -
+ +
’ The various fractions were tested for their ability to inhibit anti-Thy1 and W3/ 13 antibody binding to thymocytes. (-) >75% of cells stained; (+) ~25% of cells stained. See Materials and Methods for details.
lymph node, spleen, and bone marrow, respectively. The contention that the MPbinding cells in peripheral lymphoid tissue were T cells was supported by the observation that spleens from neonatally thymectomized rats had reduced numbers of MP-binding cells. In the present study the selective binding to peripheral T cells but not B cells was shown by direct comparison with antibodies specific for rat T cells and B cells. In spleen and lymph node MP+ cells and sIg+ cells represent distinct, nonoverlapping populations. This was shown both by single-color additive experiments with the FACS and by dual-color immunofluorescence microscopy. In contrast, all lymph node and spleen cells binding the W3/13 monoclonal antibody, which reacts with most, if not all, peripheral T cells (3), were MP+. Similarly, all W3/25+ and OX 8+ lymph node and spleen cells, which represent nonoverlapping peripheral T-cell subsets (2, 3), were MP+. Thus, all peripheral T cells, as identified by these monoclonal antibodies, bound the MP lectin. The anatomic distribution of MP in spleen and lymph node also supported the T-cell specificity. The thymus-dependent areas, the paracortex in lymph node and periarteriolar lymphatic sheath in spleen (12), were stained by the lectin, whereas the B-cell areas, the lymphoid follicles and germinal centers (12), in both tissues were not. Cells at differing stages within the T-cell developmental lineage were also MP+. Virtually all thymocytes bound the MP lectin which corroborates the findings of Jones and Feldman (5). Also, no differences in fluorescence intensity were seen between cortex and medulla of frozen thymus sections. Interestingly, approximately 20-25s of bone marrow cells containing terminal deoxynucleotidyl transferase (TdT) were MP+ and W3/13+. TdT is restricted to cortical thymocytes (13) and to a small subset of “null” cells in bone marrow and prepubertal spleen (14). Inasmuch as TdT+ bone marrow cells are present in normal proportions in congenitally athymic mice and neonatally thymectomized rats (14) and they can be induced to express thymus-specific cell surface antigens by thymic humoral factors (15) bone marrow TdT+ cells are believed to be thymocyte progenitors. The significance of a subset of TdT+ bone marrow cells being MPf, W3/ 13+ remains to be established. It seems unlikely that these are thymus-derived cells circulating from thymus to bone marrow (16) since they are W3/25-, OX 8- and all thymocytes as well as peripheral T cells are W3/25+ and/or OX 8+ (3). It is more tempting to speculate that the MP+, TdT+ bone marrow cells represent the most
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mature population of prothymocytes immediately prior to their emigration from bone marrow to thymus. This is reminiscent of thymosin induction studies by Goldschneider et al. in the mouse, in which “early” and “late” TdT+ prothymocytes were postulated (15). Both the late TdT+ cells in mouse bone marrow and the MP+, W3/ 13+ TdT+ rat bone marrow cells may serve as the immediate precursors of TdT+ thymocytes. It has been shown previously that thymic-dependent Gross virus (RAGV)-induced lymphoma leukemias in the rat may have a prethymic origin (6). These TdT+ neoplastic thymocytes contained a subband of the LDH-5 isozyme (LDH5’) that was not detected in normal thymocytes but which was present in fractions of bone marrow and prepubertal spleen cells enriched for TdT+ cells. Since many of the leukemic cells have the same phenotype as the minor population of TdT+ bone marrow cells described in the present study, it will be clearly of interest to determine if the MP+, W3/13+ TdTf bone marrow cells contain the LDH-5’. If so, it is possible that such cells are the targets of leukemic transformation by the RAGV. In a previous study, Thy1 was demonstrated on a minority of peripheral T cells as defined by the heteroantiserum ALSr (4). In the present study, similar proportions of Thyl+ T cells were found using both the W3/13 antibody and the MP lectin to identify T cells. In both studies there was a higher proportion of Thyl+ T cells in spleen than in lymph node. The Thy+ T cells were not found exclusively in either the W3/25+ or OX 8+ T-cell subset. However, a higher proportion of OX 8+ cells were Thyl+ than were W3/25+ cells. Thus, four phenotypic subsets of rat peripheral T cells can be described: (1) MP+, W3/25+, OX 8-, Thyl-; (2) MP+, W3/25-, OX 8+, Thyl-; (3) MP+, W3/25+, OX 8-, Thyl’; (4) MP+, W3/ 25-, OX 8+, Thyl’. Cells which bind W3/25 antibody exhibit helper cell activity, GVH activity, and mixed lymphocyte responsiveness (2, 3), whereas OX 8 binding cells mediate allogeneic suppression (2, 3), and have cytotoxic activity (10). Whether the four phenotypic subsets of rat peripheral T cells described above represent functionally distinct populations or whether the Thyl+ T cells are simply more recently derived from the thymus remains to be established. However, Thyl+ T cells in the rat have been reported to exhibit suppressor cell activity (17). Thus, it is tempting to speculate that functional T-cell subpopulations may be described by these four cell surface phenotypes. The specificity of binding of the MP lectin has been described previously (18). The oligosaccharides, melibiose (a disaccharide of D-galactopyranose and D-ghcase) and stachyose (a tetrasaccharide of 2-D-galactose residues, D-glucose and Dfructose), and the monosaccharide N-acetyl-galactosamine were the most potent inhibitors found. All derivatives of D-galactose exhibited significant inhibition of MP binding whereas glucose and its derivatives as well as a number of other monoand oligosaccharides were not inhibitory. The chemical composition of the W3/ 13 antigen has been described recently (19). Carbohydrate comprises 60% of the molecule and consists almost exclusively of galactose, galactosamine, and sialic acid. This carbohydrate composition is distinct from that of other known cell surface glycoproteins such as the Thy1 and leukocyte-common glycoproteins ( 19). Given the distinct carbohydrate composition of the W3/ 13 antigen and the binding specificity of the MP lectin, it is not surprising that the thymocyte glycoproteins
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which were isolated by MP lectin affinity chromatography are able to inhibit the W3/13 antibody but not the anti-Thy1 antibody. Antibodies to the Thy1 antigen were run as a control since the Thy1 carbohydrate composition is distinct from that of the W3/ 13 glycoprotein and Thy1 would be less likely to bind to the MP column. It might be argued that the repeated washing of the MP column during the elution process resulted in the nonspecific “leakage” of lectin or lectin-glycoprotein complexes. This possibility was tested by substituting glucose for melibiose during the elution process. The resulting “eluate” did not inhibit either W3/13 or anti-Thy1 antibodies. An additional control, which was not done for logistical reasons, would have been to pass a solubilized B-cell glycoprotein preparation through a MP lectin column and to test the unbound and the melibiose-eluted fractions for their ability to inhibit W3/13. Nevertheless, the present results indicate that the W3/13 glycoprotein binds to the MP lectin. However, it is not known whether the MP lectin is binding exclusively to the W3/13 glycoprotein or simply that the W3/13 glycoprotein is one of a number of glycoproteins which binds to MP. Studies are in progress to identify the cell surface molecule(s) which are bound by the MP lectin. ACKNOWLEDGMENTS The author gratefully acknowledges the excellent technical assistance of Maria Gionfriddo and the excellent assistance of Mrs. Barbara Hoekman in the preparation of the manuscript.
REFERENCES 1. Standring, R., McMaster, W. R., Sunderland, C. A., and Williams, A. F., Eur. J. Zmmunol. 8, 832, 1978. 2. Brideau, R. J., Carter, P. B., McMaster, W. R., Mason, D. W., and Williams, A. F., Eur. .I. Immunol. 10, 609, 1980. 3. Mason, D. W., Brideau, R. J., McMaster, W. R., Webb, M., White, R. A. H., and Williams, A. F., In “Monoclonal Antibodies” (R. H. Kennett and T. J. McKearn, Eds.), pp. 251-273. Plenum, New York, 1980. 4. Ritter, M. A., Gordon, L. K., and Goldschneider, I., J. Immunol. 121, 2463, 1978. 5. Jones, J. M., and Feldman, J. D., J. Immunol. 111, 1765, 1973. 6. Barton, R. W., Tausche, F., and Goldschneider, I., J. Immune!. 125, 2299, 1980. 7. Barton, R. W., Martinuik, F., Hirschhorn, R., and Goldschneider, I., J. Immunol. 122, 216, 1979. 8. Goldschneider, I., Gregoire, K. E., Barton, R. W., and Bollum, F. J., Proc. Nat. Acad. Sci. USA 74, 734, 1977. 9. Gregoire, K. E., Goldschneider, I., Barton, R. W., and Bollum, F. J., Proc. Nat. Acad. Sci. USA 74, 3993, 1977. 10. Woan, M., McGregor, D. D., and Goldschneider, I., J. Immunol. 127, 2330, 1981. il. Williams, A. F., Galfre, G., and Milstein, C., Cell 12, 663, 1977. 12. Goldschneider, I., and McGregor, D. D., J. Exp. Med. 138, 1443, 1973. 13. Barton, R. W., Goldschneider, I., and Bollum, F. J., J. Immunol. 116, 462, 1976. 14. Gregoire, K. E., Goldschneider, I., Barton, R. W., and Bollum, F. J., J. Immunol. 123, 1347, 1979. 15. Goldschneider, I., Ahmed, A., Bollum, F. J., and Goldstein, A. L., Proc. Nut. Acud. Sci. USA 78, 2469, 1981. 16. Order, S. E., and Waksman, B. H., Transplantation 8, 783, 1969. 17. Goldschneider, I., and Bollum, F. J., In “4th International Congress of Immunology (Abstracts), Paris, 1980.” 18. Cawley, L. P., Jones, J. M., and Teresa, G. W., Transfusion 7, 343, 1967. 19. Brown, W. R. A., Barclay, A. N., Sunderland, C. A., and Williams, A. F., Nature (London) 289, 456, 1981.