Proc. Nati. Acad. Sd. USA Vol. 74, No. 2, pp. 734-738, February 1977 Immunology Demonstration of terminal deoxynucleotidyl transferase in thymocytes by immunofluorescence (cortical thymocytes/medullary thymocytes/lymph node lymphocytes/antiserum to terminal transferase) IRVING GOLDSCHNEIDER*, KATHLEEN E. GREGOIRE*, RANDALL W. BARTON*, AND F. J. BOLLUMt * Department of Pathology, University of Connecticut Health Center, Farmington, Conn. 06032; and t Department of Biochemistry, University of Kentucky Medical Center, Lexington, Ky. 40506 Communicated by R. B. Setlow, December 9, 1976

ABSTRACT The cellular and subcellular distribution of Antisera. Two lots of rabbit antisera (no. 48 and no. 53) terminal deoxynucleotidyl transferase (DNA nucleotidylexo- prepared against glutaraldehyde cross-linked homogeneous calf transferase; nucleosidetriphosphate:DNA deoxynucleoti- dylexotransferase, EC 2.7.7.31) in thymocytes and peripheral transferase were used (13). F(ab')2 fragments of these antisera lymphocytes from rat, mouse, and calf was studied by immu- were prepared from purified IgG fractions by controlled pepsin nofluorescence using rabbit antiserum to homogeneous trans- digestion (cf. ref. 15). Crude IgG isolated by (NH4)2SO4 frac- ferase from calf. Terminal transferase was readily detected in tionation of serum was purified on Sephadex G-200. The IgG approximately 75% of cortical thymocytes, but not in medullary fraction from Sephadex G-200 was concentrated to 20 mg/ml, thymocytes or lymph node lymphocytes. The enzyme appeared adjusted to pH 4.5 (sodium acetate buffer), and digested with to be present predominantly in the cytoplasm of positive thy- pepsin (Worthington) per 100 mocytes in ethanol-fixed cell smears and frozen sections. The 2 mg of twice-crystallized hog reactivity of anti-terminal-transferase for thymocytes could be mg of IgG protein. Progress of the digestion was followed by neutralized with purified calf enzyme. Results of experiments analyzing aliquots of the digestion mixture on sodium dodecyl in which thymocytes were separated on 7-step discontinuous sulfate/polyacrylamide slab gels and observing the loss of Ficoll density gradients suggested that cortical thymocytes are staining intensity in the heavy chain region. Aliquots of reaction heterogeneous with respect to terminal deoxynucleotidyl mixture were also analyzed by microimmunoelectrophoresis transferase content. in 1% agarose (50 mM sodium diethylbarbiturate buffer, pH Terminal deoxynucleotidyl transferase (DNA nucleotidylex- 8.4) and testing with goat antiserum to rabbit IgG and goat otransferase; nucleosidetriphosphate:DNA deoxynucleoti- antiserum to rabbit Fc (gift of T. R. Roszman). In this analysis EC is an that catalyzes the loss of Fc reactivity and change in mobility of the.IgG re- dylexotransferase, 2.7.7.31) enzyme activity signifies conversion to F(ab')2. Generally speaking, 16 synthesis of deoxynucleotide sequences without template di- hr of digestion followed by addition of another 2 mg of pepsin rection (1) and that appears to be normally restricted to thymus and a further 6 hr digestion was required to obtain complete and bone marrow (2-6). This transferase has also been found conversion to F(ab')2. in neoplastic cells from human patients with acute lympho- The pepsin digests were fractionated on Sephadex G-200 and blastic leukemia and in several thymus-derived (T) cell leuke- the F(ab')2 obtained was dialyzed against 10 mM potassium mia and lymphoma cell lines from man and mouse (7-10, t). phosphate at pH 7.2. The dialyzed F(ab')2 was then passed Transferase has not been identified in normal or neoplastic cells through a 1 X 10 cm column of Sephadex A-25 and washed expressing bone-marrow-derived (B) cell characteristics. through with 10 mM phosphate buffer. The nonadsorbed Previous functional and antigenic analyses of thymocyte fractions containing F(ab')2 and antiterminal-transferase ac- suspensions separated on discontinuous density gradients sug- tivity were lyophilized or used directly. gested that cortical thymocytes were transferase-positive (4, Aliquots of the antiterminal-transferase were diluted 1:5 in 11, 12). Medullary thymocytes were judged to be transferase- Dulbecco's phosphate-buffered saline (containing 20 mM negative (12). We have now directly confirmed these impres- NaN3) and absorbed twice (4, 20 min) with equal volumes of sions by using rabbit antiserum to homogeneous calf thymus packed rat erythrocytes. Additional absorptions were per- transferase (13) to demonstrate transferase within the cytoplasm formed with viable rat lymph node cells or thymocytes or with of cortical thymocytes from rat, mouse, and calf. purified calf transferase as indicated under Results. Absorption METHODS with transferase (gift of L. M. S. Chang) was performed by adding 1000 units (1 unit catalyzes polymerization of 1 nmol Lymphoid Tissues. Thymus, cervical lymph nodes, and of nucleotide per/hr under standard assay conditions) of ho- blood were obtained from 8- to 12-week-old male and female mogeneous transferase (10 ul) to 100 Ml of clarified antiserum (Lewis X DA)F1 strain hybrid rats and 8-week-old C3H and and then diluting to 500 MAl with phosphate-buffered saline. The AKR strain mice. Bovine thymus and bronchial lymph nodes mixture was allowed to stand at 40 for 4 hr and was again were obtained from a freshly slaughtered, 6-month-old female clarified at 100,000 X g for 60 min. Dilutions of the supernatant Holstein calf. Frozen sections and cell suspensions were pre- fraction were used directly as "transferase-absorbed" antise- pared as described previously (14). In some experiments thy- rum. mocytes were separated on a 7-step discontinuous Ficoll density Rabbit antiserum to the rat bone marrow lymphocyte antigen gradient (8-20%, wt/vol) (12) before immunofluorescent (anti-BMLA) was used to identify cortical thymocytes (16). staining. Lewis strain rat antiserum to DA strain rat histocompatibility antigens (Le anti-DA) was used to identify medullary thymo- Abbreviations: transferase, terminal deoxynucleotidyl transferase; T cells, thymus-derived cells; B cells, bone-marrow-derived cells; FITC, cytes (12). fluorescein isothiocyanate. Fluorescein-conjugated rabbit IgG against rat IgG (FITC- f M. S. Coleman, M. F. Greenwood, J. J. Hutton, F. J. Bollum, B. anti-rat IgG), fluorescein-conjugated goat IgG against rabbit Lampkin, and P. Holland, J. Clin. Invest., in press. IgG (FITC-anti-rabbit IgG), and goat IgG against rabbit IgG 734 Downloaded by guest on September 29, 2021 Immunology: Goldschneider et al. Proc. Natl. Acad. Sci. USA 74 (1977) 735 were obtained from Cappel Laboratories, Downingtown, Pa. Immunofluorescence. Suspensions of living cells and frozen sections of fresh tissues were processed for indirect immu- nofluorescence as described previously (14). Appropriately absorbed preimmunization rabbit serum and IgG and F(ab')2 fractions of normal rabbit serum were used as controls for the corresponding antiterminal-transferase preparations. Cell smears were made on alcohol-cleaned glass slides, using a 3% suspension (vol/vol) of packed cells in Tyrode's solution con- taining 50% (vol/vol) fetal calf serum. The frozen sections and cell smears were fixed in 95% ethanol (40, 5 min) and were washed twice in phosphate-buffered saline prior to reacting with antiserum. Absolute methanol was also a suitable fixative. Acetone, 2% (vol/vol) buffered formalin, and mixtures of ethanol/formalin/acetic acid were less satisfactory because they did not prevent diffusion of transferase from the cells. Higher concentrations of formalin destroyed the antigenicity of transferase, whereas glutaraldehyde (0.5-2.5%, vol/vol) in- troduced unsatisfactory levels of autofluorescence. Enzyme Assay. Terminal deoxynucleotidyl transferase en- zymatic activity in sonicated cell suspensions was assayed as described previously (12).

RESULTS Distribution of transferase-positive cells in thymus sections Frozen sections of rat, mouse, and calf thymus were exposed to serial 2-fold dilutions of rabbit antiterminal-transferase serum, and to the IgG and F(ab')2 fractions thereof, and were FIG. 1. Frozen section of normal rat thymus, fixed in ethanol, processed for indirect immunofluorescence. Although the exposed to rabbit F(ab')2 antiterminal-transferase, and developed pattern and intensity of specific fluorescence was not altered with FITC-anti-rabbit IgG, X256. Exposure 3 min (Polaroid type by fractionation of the antiterminal-transferase serum, there 57 black and white film, ASA 3000). Thymocytes in cortex (C) fluo- was a marked reduction in the level of nonspecific fluorescence resce brightly. The pattern of fluorescence is consistent with cyto- in sections stained with the F(ab')2 antiterminal-transferase. plasmic and/or cell-surface staining. There is no detectable nuclear staining. Thymocytes and epithelial cells in medulla (M) do not A similar reduction in nonspecific staining occurred when the fluoresce. Connective tissue elements in medulla and thymus capsule antiterminal-transferase preparations were absorbed with rat (arrow) fluoresce nonspecifically. Macrophages in region of cortico- erythrocytes and viable lymph node cells or thymocytes. In- medullary junction (broken line) show bright yellow autofluores- asmuch as these absorptions did not significantly affect anti- cence. terminal-transferase activity when tested by enzyme neutral- ization assay (13), they were performed routinely. As shown in Fig. 1, rat cortical thymocytes fluoresced Cytological localization of transferase brightly with antiterminal-transferase (titer 1:80), whereas F(ab')2 antiterminal-transferase (diluted 1:5) was incubated medullary thymocytes did not fluoresce detectably (titer <1: with suspensions of viable rat thymocytes and lymph node cells 10). This was true for rat, mouse, and calf thymus sections. The and developed for immunofluorescence. There was no de- ring-like pattern of fluorescence seen at the periphery of the tectable surface staining of either cell type. The titer of the cell could represent surface and/or cytoplasmic staining, be- antiterminal-transferase was not significantly reduced by ab- cause thymocytes have only a narrow rim of cytoplasm. There sorption with viable thymocytes or lymph node cells, as de- was no detectable nuclear staining in these preparations. It was termined by its ability to neutralize transferase activity (13) and our impression that thymocytes in the deepest part of the cortex to react with frozen sections of thymus. (i.e., near the cortico-medullary junction) fluoresced less in- F(ab')2 antiterminal-transferase did react with approximately tensely than thymocytes elsewhere in the cortex, and that they 64% of total thymocytes in ethanol-fixed cell smears (Fig. 2a). were admixed with transferase-negative cells. Epithelial cells The cytoplasm of positive cells fluoresced intensely, whereas in the thymus medulla were transferase-negative. Epithelial the nucleus generally appeared to be negative. An occasional cells in the thymus cortex were obscured by fluorescing thy- large thymocyte displayed very weak staining in the region of mocytes and could not be evaluated. the nucleus. Ethanol-fixed lymph node cells showed neither The specificity of the reaction of antiterminal-transferase cytoplasmic nor nuclear fluorescence (Fig. 2b). with cortical thymocytes was confirmed by results of the fol- lowing experiments: (i) preimmunization rabbit serum that had Heterogeneity of cortical thymocytes been absorbed with erythrocytes and lymph node cells did not Suspensions of rat thymocytes were separated on 7-step Ficoll react with cortical thymocytes; (ii) similarly absorbed antiter- density gradients. The percentages of transferase-positive cells minal-transferase did not react with frozen sections of cervical, and cortical thymocytes were measured by immunofluores- mesenteric, or bronchial lymph nodes; and (iii) reactivity of cence in each fraction (Fig. 3). Approximately 75% of total antiterminal-transferase for cortical thymocytes in sections was cortical thymocytes were transferase-positive. However, this removed by absorption with purified calf transferase. value varied considerably in individual fractions of the gradient. Downloaded by guest on September 29, 2021 736 Immunology: Goldschneider et al. Proc. Natt. Acad. Sci. USA 74 (1977)

60- LUJ

0 CL UJ LU

20

1 2 3 4 5 6 7 FRACTION NUMBER FIG. 3. Analysis of rat thymocyte gradient fractions with antisera specific for cortical thymocytes and terminal deoxynucleotidyl transferase. 0, % cortical thymocytes; *, % transferase-positive cells. Each point represents the mean of four experiments. See Methods for description of antisera and procedure for immunofluorescence.

However, there was no apparent difference in intensity of fluorescence between transferase-positive cells in fractions 5 and 7, despite the marked differences in transferase specific activity (27.3 versus 9.3 transferase units/108 transferase-pos- itive cells, respectively). FIG. 2. Smears of normal rat thymocytes and cervical lymph node cells, fixed in ethanol, exposed t& F(ab')2 antiterminal-transferase DISCUSSION and developed with FITC-anti-rabbit IgG, X525. Exposure 5 min (Kodak Tri-X Pan 35 mm film, ASA 400). (a) Thymocytes. There is The physiological function of terminal deoxynucleotidyl intense cytoplasmic fluorescence of a majority of the cells in the smear transferase is not known. The restriction of distribution of (negative cells are indicated by arrows). There is no detectable nuclear transferase to thymus and bone marrow has prompted specu- staining beyond background levels seen in F(ab')2 normal rabbit lation that it is involved in the functional differentiation of T serum controls. (b) Lymph node cells. The cells appear as barely cells, perhaps in immunological memory or diversity (1, 5, 17, visible ghosts. There is no detectable nuclear or cytoplasmic fluo- 18). Whatever the merits of these proposals, it seems reasonable rescence beyond background levels seen in controls (also compare with thymocytes). Bright particulate material is precipitated fluorescein to anticipate that studies of the cellular and subcellular distri- conjugate. bution of transferase may provide important insights both into the pathways of T cell development, and into the possible role(s) of transferase in this development. The availability of an anti- For example, more than 95% of cortical thymocytes in fractions serum specific for transferase (13) promises to greatly expedite 5 and 6 were transferase-positive, whereas less than 10% of such studies. cortical thymocytes in fractions 1 and 2 were transferase-pos- In.the present report we have directly confirmed our previous itive. Moreover, it was our impression that many of the trans- findings (11, 12) that transferase-positive thymocytes in the ferase-positive thymocytes in fractions 3 and 4 fluoresced less adult rat are restricted to the thymus cortex. We have extended intensely than the transferase-positive thymocytes in fractions these observations to include the thymus of the mouse and calf. 5-7. This suggested that heterogeneity exists among transfer- However, not all cortical thymocytes were found to be trans- ase-positive thymocytes with respect to transferase content. ferase-positive, and not all transferase-positive cells appeared In an attempt to document the latter point, transferase spe- to contain equal amounts of enzymatically active transferase. cific activity (measured enzymatically) was calculated as a This heterogeneity was demonstrated by analysis of suspensions function of the percent of transferase-positive cells in each of thymocytes separated on Ficoll density gradients The results fraction (determined by immunofluorescence). The results indicate that the majority of high- and low-density cortical presented in Table 1 confirm our impression that transferase- thymocytes are transferase-negative, whereas almost all me- positive cells in fractions 3 and 4 fluoresced less intensely with dium-density cortical thymocytes are transferase-positive (Fig. antiterminal-transferase than did cells in other fractions. 3). Moreover, transferase-positive cells in low-density fractions Downloaded by guest on September 29, 2021 immunology: Goldschneider et al. Proc. Natl. Acad. Sci. USA 74 (1977) 737 Table 1. Specific activity in thymocyte gradient fractions* Transferase Transferase Percent trans- units/108 trans- Intensity of Fraction no. units/10' cellst ferase-positive cellst ferase-positive cells fluorescence 1 (top) 1.0 3 ?§ ?§ 2 1.5 7 ?§ ?§ 3 2.3 15 15.3 Low 4 5.6 30 17.0 Low 5 18.0 66 27.3 High 6 16.3 77 21.2 High 7 (bottom) 4.1 44 9.3 High * Means of two experiments. t Determined by enzymatic analysis of cell extracts (see Methods). I Determined by immunofluorescence with rabbit F(ab')2 antiterminal-transferase (see Methods). § Insufficient transferase-positive cells to evaluate fluorescence and enzyme content.

appear to contain less enzyme than those in medium-density 2. Chang, L. M. S. (1971) "Development of terminal deoxynu- fractions, as evidenced by weak immunofluorescence with cleotidyl transferase activity in embryonic calf thymus gland," Biochem. Biophys. Res. Commun. 44, 124-131. antiterminal-transferase and low transferase specific activity 3. Coleman, M. S., Hutton, J. J., De Simone, P. & Bollum, F. J. (1974) (compare fractions 3 and 5, Table 1). In contrast, some trans- "Terminal deoxynucleotidyl transferase in human leukemia," ferase-positive cells in the high density fraction may contain Proc. Natl. Acad. Sci. USA 71, 4404-4408. antigenically intact but enzymatically inactive enzyme. Such 4. Kung, P. C., Silverstone, A. E., McCaffrey, R. P. & Baltimore, cells appeared to fluoresce as intensely with antiterminal- D. (1975) "Murine terminal deoxynucleotidyl transferase: Cel- transferase as did medium-density thymocytes, but had only lular distribution and response to cortisone," J. Exp. Med. 141, one-third the transferase specific activity (compare fractions 855-865. 7 and 5, Table 1). 5. Bollum, F. J. (1975) "Terminal deoxynucleotidyl transferase Results of the present study indicate that transferase in cor- (TdT) in dexamethasone-treated rat tissues," Fed. Proc. 34,- tical thymocytes is located predominantly within the cytoplasm. 494. 6. Barr, R. D., Sarin, P. S. & Perry, S. M. (1976) "Terminal trans- There was no detectable transferase on the surface of viable ferase in human bone-marrow lymphocytes," Lancet i, 508- thymocytes. A nuclear pattern of fluorescence with antiter- 509. minal-transferase was only occasionally observed. This is in 7. McCaffrey, R., Smoler, D. F. & Baltimore, D. (1973) "Terminal substantial agreement with the study of Chang (2), which deoxynucleotidyl transferase in a case of childhood acute lym- showed that the bulk of transferase activity was present in the phoblastic leukemia," Proc. Natl. Acad. Sci. USA 70, 521- soluble (nonnuclear) fraction of disrupted thymocytes. How- 525. ever, that study also reported that approximately one-third of 8. McCaffrey, R., Harrison, T. A., Parkman, R. & Baltimore, D. transferase activity in calf thymus was associated with the nu- (1975) "Terminal deoxynucleotidyt transferase activity in human clear fraction, and that transferase activity was present in iso- leukemic cells and in normal human thymocytes," N. Engl. J. lated rat thymocyte nuclei. The question of the exact intra- Med. 292, 775-780. 9. Gallo, R. C. (1975) "Terminal transferase and leukemia," New cellular distribution of transferase must therefore await more Engl. J. Med. 292,804-805. definitive immunocytological studies, preferably at the ultra- 10. Srivastava, B. I. S. & Minowada, J. (1973) "Terminal deoxynu- structural level. cleotidyl transferase in a cell line (Molt-4) derived from the pe- Our inability to demonstrate transferase in medullary thy- ripheral blood of a patient with acute lymphoblastic leukemia," mocytes or in lymph node by fluorescence is consistent with the Biochem. Biophys. Res. Commun. 51,529-535. findings of earlier biochemical studies (2, 11). Taken together, 11. Coleman, M. S., Hutton, J. J. & Bollum, F. J. (1974) "Terminal these results suggest that transferase does not exist in an enzy- deoxynucleotidyl transferase and DNA polymerase in classes of matically active or inactive form in recirculating T cells or in cells from rat thymus," Biochem. Biophys. Res. Commun. 58, B cells. Moreover, it raises the possibility that transferase-pos- 1104-1109. 12. Barton, R., Goldschneider, I. & Bollum, F. J. (1976) itive cells and their may not "The distri- descendants normally leave the bution of terminal deoxynucleotidyl transferase (TdT) among thymus. Thus, it will be important to determine if medullary subsets of thymocytes in the rat," J. Immunol. 116, 462-468. thymocytes and spleen-seeking thymocytes (19, 20) are derived 13. Bollum, F. J. (1975) "Antibody to terminal deoxynucleotidyl from transferase-positive progenitors in thymus cortex. These transferase," Proc. Nat. Acad. Sci. USA 72, 4119-4122. considerations have been discussed elsewhere (12, 16). 14. Goldschneider, I. & McGregor, D. D. (1973) "Anatomical dis- tribution of T and B lymphocytes in the rat: Development of lymphocyte-specific antisera," J. Exp. Med. 138, 1443-1465. This work was supported by Grant AI-09649 from the National In- 15. Stanworth, D. R. & Turner, M. W. (1973) "Immunochemical stitute of Allergy and Infectious Diseases and CA-08487 from the analysis of immunoglobulins and their subunits," in Handbook National Cancer Institute. R.W.B. is the recipient of Public Health of Experimental Immunology, ed. Weir, D. W. (Blackwells, Service Research Fellowship AI-05037. We are indebted to Dr. L. M. Oxford), Vol I, pp. 10-16. S. Chang for a gift of homogeneous transferase, and to Dr. T. R. 16. Goldschneider, I. (1976) "Antigenic relationship between bone Roszman for goat anti-rabbit Fc serum. marrow lymphocytes, cortical thymocytes and a subpopulation of peripheral T cells in the rat: Description of a bone marrow lymphocyte antigen," Cell. Immunol. 24, 289-307. 1. Bollum, F. J. (1974) "Terminal deoxynucleotidyl transferase," 17. Baltimore, D. (1974) "Is terminal deoxynucleotidyl transferase in The Enzymes, ed. Boyer, P. D. (Academic Press, Inc., New a somatic mutagen in lymphocytes?" Nature 248,409-410. York), Vol. 10, pp. 145-171. 18. Bollum, F. J. (1975) "Terminal deoxynucleotidyl transferase; Downloaded by guest on September 29, 2021 738 Immunology: Goldschneider et al. Proc. Nat!. Acad. Sci. USA 74 (1977)

source of immunological diversity?" Karl August Forster Lec- Immunofluorescence and 3H TdR labelling studies," Adv. Exp. tures, Verlag Akad. Wiss. Lit. Mainz (Steiner Verlag, Wiesbaden), Med. Biol. 12,113-118. Vol. 14. 20. Zatz, M. M. & Lance, E. M. (1970) "The distribution of chromium 19. Chanana, A. D., Cronkite, E. P., Joel, D. D., Williams, R. M. & 51-labelled lymphoid cells in the mouse. A survey of anatomical Waksman, B. H. (1971) "Migration of thymic lymphocytes: compartments," Cell. Immunol. 1, 3-17. Downloaded by guest on September 29, 2021