Clin. exp. Immunol. (1976) 26, 501-504.
BRIEF COMMUNICATION
Spontaneous rosette formation by lymphocytes DIFFERENTIAL EFFECT OF CYTOCHALASIN B ON THYMOCYTES AND THYMUS-RELATED PERIPHERAL BLOOD LYMPHOCYTES IN THE GUINEA-PIG T. RADASZKIEWICZ, D. KOTZ & H. DENK Department of Pathology, University of Vienna School of Medicine, Vienna, Austria
(Received 12 January 1976)
SUMMARY
In the present investigation the differences in the capacity of thymocytes (Th) and thymusrelated peripheral blood lymphocytes (TBL) of the guinea-pig to form spontaneous rosettes with rabbit erythrocytes (RRBC) were further evaluated. Cytochalasin B (CB) inhibited TBL rosette formation but was largely ineffective with respect to Th. This suggests that membrane modulation and microfilament function are requirements for the formation of TBL but not of Th rosettes. A certain critical RRBC receptor density on the lymphocyte may be necessary for a stable rosette. Our results suggest that different RRBC receptor densities on Th and TBL account for the differences in rosette forming capacity of these two cell types. In native Th, the receptor density may be high enough to warrant stable rosette formation. In TBL, however, active membrane movement and perhaps clustering of receptors may be necessary to achieve the local critical receptor density as prerequisite of a stable rosette.
INTRODUCTION Spontaneous rosette formation between guinea-pig lymphoid cells and rabbit erythrocytes is considered a T-cell marker (Brain, Gordon & Willetts, 1970; Wilson & Coombs, 1973; Stadecker, Bishop & Wortis, 1973; Radaszkiewicz & Denk, 1974, 1975) similar to the spontaneous rosetting of sheep etythrocytes around human T cells (Bach, 1973; Coombs et al., 1970; Lay et al., 1971; Jondal, Holm & Wigzell, 1972; Fr0land, 1972; Wybran, Carr & Fudenberg, 1972). In the guinea-pig model, conflicting results have been reported on the metabolic requirements for this reaction (Wilson & Coombs, 1973; Stadecker et al., 1973). In a previous paper it was shown that significant differences exist between thymocytes (Th) and thymus-dependent peripheral blood lymphocytes (TBL) in this respect-Th were able to rosette with rabbit erythrocytes (RRBC) in the life as well as in the metabolically inhibited state whereas TBL lost their capacity for rosette formation if metabolically inhibited (Radaszkiewicz & Denk, 1975). Heating prevented resetting in both cell types (Baxley et al., 1973; Radaszkiewicz & Denk, 1975) presumably by release of the erythrocyte receptor (Mendes, Saraiva & Santos, 1975). The basic mechanism of the rosetting phenomenon is as yet poorly understood. The involvement of microfilament action was postulated from the inhibition exerted by cytochalasin B (CB) (Kersey, Hom & Buttrick, 1974; Cohnen, Fischer & Brittinger, 1975). The present experiments were designed to study the affect of CB on the rosette-forming capacity of Th and TBL. Correspondence: Dr H. Denk, Department of Pathology, University of Vienna School of Medicine, Spitalgasse 4, A-1090 Vienna, Austria.
501
T. Radaszkiewicz, D. Kotz L H. Denk
502
MATERIALS AND METHODS Guinea-pig TBL, Th and RRBC were prepared and the rosetting reaction was performed as described previously (Radaszkiewicz & Denk, 1975). In each experiment pooled TBL and Th of four to five guinea-pigs (randomly bred animals) were used. Viability of the lymphoid cells was determined by trypan blue exclusion and was found to be > 98%. Contamination of the lymphocytes with granulocytes was < I%. To study the CB-effect on rosette formation, lymphocytes (15 x 106 cells/ml) were incubated for 60 min at 370C in RPMI 1640 medium (GIBCO, Glasgow) containing CB in a final concentration of 2, 10, 20 and 40 #g/ml. After incubation, the cells were centrifuged and washed three times with RPMI 1640 medium at 40C. Rosettes were prepared immediately afterwards as described (Radaszkiewicz & Denk, 1975). CB was prepared as a stock solution in dimethylsulphoxide (DMSO) at a concentration of 5 mg CB in 0 5 ml DMSO. This stock solution was diluted with phosphate-buffered saline (PBS), pH 7 4, to double final CB-concentration and mixed with an equal amount of the lymphocyte suspension in RPMI 1640 medium (30 x 106 cells/ml). DMSO alone in identical dilution was used in control assays. In addition, for comparison the effect of NaCN in a concentration of 15 mm was tested on TBL and Th as described (Radaszkiewicz & Denk, 1975). To further evaluate the metabolic requirements for rosette formation and rosette stability additional experiments were performed in order to assess whether metabolic integrity of the lymphocyte is required to maintain an already formed rosette. For this purpose, NaCN (15 mm final concentration) was added to TBLRRBC rosette.
RESULTS CB proved to be a potent inhibitor of TBL-rosette formation (Table 1). Even in the low concentration of 2 pg/ml the rosette forming capacity was decreased to 5000 of the controls, 10 pug/ml showed already near maximal inhibition. In higher concentrations CB was as effective an inhibitor as NaCN. The solvent itself (DMSO) was ineffective. Th rosette formation was largely insensitive to CB in concentrations up to 20 pg/ml; at 40 ug/ml, however, some inhibition was observed (Table 1). Although NaCN in the concentration used (15 mM) inhibited TBL-rosette formation significantly as seen in Table 1, this drug was ineffective if added to the already formed rosette. For example, before addition of NaCN 21% rosette-forming TBL were found and after NaCN the value was at 23%4. TABLE 1. Effect of CB and NaCN on rosette formation by Th and TBL. The results of one representative experiment are listed (the experiments were repeated at least three times)
Percentage
rosette-forming cells*
Lymphocytes
NaCN
Cytochalasin B (pug/ml) 2
Tht TBLt
10
20
40
15 mM
76
80
78
37
78
(74)
(77)
(76)
(67)
(78)
9 (21)
7 (23)
6 (20)
5
5
(21)
(25)
* Numbers in parentheses are the percentages of the respective controls (DMSO in case of CB). t Pooled cells of four guinea-pigs.
DISCUSSION Metabolic activity is not an absolute requirement for spontaneous rosette formation between thymusrelated lymphocytes and RRBC in the guinea-pig model (Wilson & Coombs, 1973; Radaszkiewicz & Denk, 1975). Guinea-pig Th formed spontaneous rosettes in the live as well as in the dead state (Radaszkiewicz & Denk, 1974, 1975) whereas TBL did not if metabolically inhibited (Stadecker et al.,
Guinea-pig T cells-rabbit red cell rosettes
503
1973; Radaszkiewicz & Denk, 1975). Once established, however, TBL rosettes were not affected by metabolic inhibitors like cyanide. This indicates that metabolic cellular processes in the lymphocytes are only necessary to initiate but not to maintain the spontaneous rosette. CB, an inhibitor of microfilament function (Wessels et al., 1971) did not affect spontaneous rosette formation of Th but inhibited TBL rosettes. The different response of Th and TBL could be due to different receptor concentrations on the lymphocyte surface whereby a certain critical receptor density may be necessary for a stable rosette. In Th a high receptor density could support rosette formation even in the dead or metabolically inhibited state. In TBL with sparse receptors achievement of a critical density could require receptor movement and clustering upon contact with RRBC-a CBsensitive process. Clustering of surface receptors on various cells is well known (Ashman, 1973; Ashman & Raff, 1973; Singer, 1974; Thiele & Stark, 1974; Loor, Block & Little, 1975). Our experimental conditions make it unlikely that TBL rosette formation requires a typical capping of RRBC receptors since Yu (1974) has shown that capping of the erythrocyte receptors on human T cells counteracts rosette formation and is even the reason for the dissociation of an already formed rosette. Furthermore, as in B cells, classical capping of the erythrocyte receptor requires incubation at 370C (Yu, 1974) rather than the 4VC used in our experiments. The hypothesis of different receptor densities on Th and TBL is in line with the findings of several authors that certain thymus specific antigens are present in a higher concentration on the Th than on the TBL surface (Aoki et al., 1969; Raff, 1970). The higher amounts of contractile proteins in association with the TBL membrane (Fagraeus, The & Biberfeld, 1973; Fagraeus, Lidman & Biberfeld, 1974) would then appear as logical consequence to compensate, by increased active membrane mobility, for the lower receptor density. However, further evaluation of this hypothesis requires direct detection of the RRBC receptor on the lymphoid cell surface. This research was supported in part by Fonds zur Forderung der wissenschaftlichen Forschung (no. 1543) and by AdolfSonnleitner-Fonds of the Austrian Academy of Sciences.
REFERENCES AOKI, T., HAMMERLING, U., HARVEN, E. DE, BoYsE, E.A. & OLD, L.J. (1969) Antigenic structure of cell surfaces. An immunoferritin study of the occurrence and topography of H-2, 9, and TL alloantigens on mouse cells. I. exp. Med. 130, 979. ASHMAN, R.F. (1973) Lymphocyte receptor movement induced by sheep erythrocyte binding. 7. Immunol. 111, 212. ASHMAN, R.F. & RAFF, M. (1973) Direct demonstration of Theta-positive antigen-binding cells, with antigeninduced movement of thymus-dependent cell receptors. .7. exp. Med. 137, 69. BACH, J.F. (1973) Evaluation of T-cells and thymic serum factors in man using the rosette technique. Transplant. Rev. 16, 196. BAXLEY, G., BIsHoP, G.B., COOPER, A.G. & WORTIs, H.H. (1973) Rosetting of human red blood cells to thymocytes and thymus-derived cells. Clin. exp. Immunol. 15, 385. BRAIN, P., GORDON, J. & WILLETTS, W.A. (1970) Rosette formation by peripheral lymphocytes. Clin. exp. Immunol. 6, 681. COHNEN, G., FisCHER, K. & BRITTINGER, G. (1975) Human T-lymphocyte rosette formation. Inhibition by Cytochalasin B. Immunology, 29, 337. COOMBS, R.R.A., GuRr-ER, B.W., WILSON, A.B., HoLm, G. & LINDGREN, B. (1970) Rosette formation between human lymphocytes and sheep red cells not involving immunoglobulin receptors. Int. Arch. Allergy, 39, 658. FAGRAEUS, A., LIDMAN, K. & BIBERFELD, (G. (1974) Re-
action of human smooth muscle antibodies with human blood lymphocytes and lymphoid cell lines. Nature (Lond.), 252, 246. FAGRAEUS, A., THE, H. & BIBERFELD, G. (1973) Reaction of human smooth muscle antibody with thymus medullary cells. Nature: New Biology, 246, 113. FRoLAND, S.S. (1972) Binding of sheep erythrocytes to human lymphocytes. A probable marker of T-lymphocytes. Scand. J. Immunol. 1, 269. JoNDAL, M., HOLM, G. & WIGzELL, H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. 5. exp. Med. 136, 207. KERsEY, J.N., HOM, D.J. & BuTTRICK, P. (1974) Human T lymphocyte receptors for sheep erythrocytes: conditions for binding including inhibition by Cytochalasin B. 5. Immunol. 112, 862. LAY, W.H., MF.NDEs, N.F., BIANco, C. & NUSSENZWEIG, V. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531. LOOR, F., BLOCK, N. & LiTTLE, J.R. (1975) Dynamics of the TL antigens on thymus and leukemia cells. Cell. Immunol. 17, 351. AMNDEs, N.F., SARAIVA, P.J. & SANTOS, O.B.O. (1975) Restorative effect of normal human serum, transfer factor and thymosin on the ability of heated human lymphocytes to form rosettes with sheep erythrocytes. Cell. Immunol. 17, 560. RADASZKIEWICZ, T. & DENK, H. (1974) Identification of
504
T. Radaszkiewicz, D. Kotz & H. Denk
thymus dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes. Cell. Immunol. 14, 303. RADASZKIEWICZ, T. & DENK, H. (1975) Rosette formation of guinea pig lymphoid cells with rabbit erythrocytesdifferences between thymocytes and peripheral blood lymphocytes. Cell. Immunol. 16, 374. RAFF, M.C. (1970) Two distinct populations of peripheral lymphocytes in mice distinguishable by immunofluorescence. Immunology, 19, 637. SINGER, S.J. (1974) Molecular biology of cellular membranes with applications to immunology. Advances in Immunology (ed. by F. J. Dixon & H. G. Kunkel), volume 19, p. 1. Academic Press, New York. STADECKER, M.J., BISHOP, G. & WORTIS, H.H. (1973) Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells. J. Immunol. 111, 1834. THIELE, H.-G. & STARK, R. (1974) Common antibody-
induced redistribution on thymocytes of strain-speciesand non-species-specific antigenic determinants shared by brain and thymocytes of mice. Immunology, 27, 807. WESSELS, N.K., SPOONER, B.S., ASH, J.F., BRADLEY, M.O. LuDuENA, M.A., TAYLOR, E.L., WRENN, J.T. & YAMADA, K.M. (1971) Microfilaments in cellular and developmental process. Contractile microfilament machinery of many cell types is reversibly inhibited by cytochalasin B. Science, 171, 135. WILSON, A.B. & COOMBS, R.R.A. (1973) Rosette formation between guinea pig lymphoid cells and rabbit erythrocytes -a possible T-cell marker. Int. Arch. Allergy, 44, 544. WYBRAN, J., CARR, M.C. & FUDENBERG, H.H. (1972) The human rosette-forming cell as a marker of a population of thymus derived cells. J. cdin. Invest. 51, 2537. Yu, D.T.Y. (1974) Human lymphocyte receptor movement induced by sheep erythrocyte binding: Effect of temperature and neuraminidase treatment. Cell. Immunol. 14, 313.
BRIEF COMMUNICATION
Spontaneous rosette formation by lymphocytes DIFFERENTIAL EFFECT OF CYTOCHALASIN B ON THYMOCYTES AND THYMUS-RELATED PERIPHERAL BLOOD LYMPHOCYTES IN THE GUINEA-PIG T. RADASZKIEWICZ, D. KOTZ & H. DENK Department of Pathology, University of Vienna School of Medicine, Vienna, Austria
(Received 12 January 1976)
SUMMARY
In the present investigation the differences in the capacity of thymocytes (Th) and thymusrelated peripheral blood lymphocytes (TBL) of the guinea-pig to form spontaneous rosettes with rabbit erythrocytes (RRBC) were further evaluated. Cytochalasin B (CB) inhibited TBL rosette formation but was largely ineffective with respect to Th. This suggests that membrane modulation and microfilament function are requirements for the formation of TBL but not of Th rosettes. A certain critical RRBC receptor density on the lymphocyte may be necessary for a stable rosette. Our results suggest that different RRBC receptor densities on Th and TBL account for the differences in rosette forming capacity of these two cell types. In native Th, the receptor density may be high enough to warrant stable rosette formation. In TBL, however, active membrane movement and perhaps clustering of receptors may be necessary to achieve the local critical receptor density as prerequisite of a stable rosette.
INTRODUCTION Spontaneous rosette formation between guinea-pig lymphoid cells and rabbit erythrocytes is considered a T-cell marker (Brain, Gordon & Willetts, 1970; Wilson & Coombs, 1973; Stadecker, Bishop & Wortis, 1973; Radaszkiewicz & Denk, 1974, 1975) similar to the spontaneous rosetting of sheep etythrocytes around human T cells (Bach, 1973; Coombs et al., 1970; Lay et al., 1971; Jondal, Holm & Wigzell, 1972; Fr0land, 1972; Wybran, Carr & Fudenberg, 1972). In the guinea-pig model, conflicting results have been reported on the metabolic requirements for this reaction (Wilson & Coombs, 1973; Stadecker et al., 1973). In a previous paper it was shown that significant differences exist between thymocytes (Th) and thymus-dependent peripheral blood lymphocytes (TBL) in this respect-Th were able to rosette with rabbit erythrocytes (RRBC) in the life as well as in the metabolically inhibited state whereas TBL lost their capacity for rosette formation if metabolically inhibited (Radaszkiewicz & Denk, 1975). Heating prevented resetting in both cell types (Baxley et al., 1973; Radaszkiewicz & Denk, 1975) presumably by release of the erythrocyte receptor (Mendes, Saraiva & Santos, 1975). The basic mechanism of the rosetting phenomenon is as yet poorly understood. The involvement of microfilament action was postulated from the inhibition exerted by cytochalasin B (CB) (Kersey, Hom & Buttrick, 1974; Cohnen, Fischer & Brittinger, 1975). The present experiments were designed to study the affect of CB on the rosette-forming capacity of Th and TBL. Correspondence: Dr H. Denk, Department of Pathology, University of Vienna School of Medicine, Spitalgasse 4, A-1090 Vienna, Austria.
501
T. Radaszkiewicz, D. Kotz L H. Denk
502
MATERIALS AND METHODS Guinea-pig TBL, Th and RRBC were prepared and the rosetting reaction was performed as described previously (Radaszkiewicz & Denk, 1975). In each experiment pooled TBL and Th of four to five guinea-pigs (randomly bred animals) were used. Viability of the lymphoid cells was determined by trypan blue exclusion and was found to be > 98%. Contamination of the lymphocytes with granulocytes was < I%. To study the CB-effect on rosette formation, lymphocytes (15 x 106 cells/ml) were incubated for 60 min at 370C in RPMI 1640 medium (GIBCO, Glasgow) containing CB in a final concentration of 2, 10, 20 and 40 #g/ml. After incubation, the cells were centrifuged and washed three times with RPMI 1640 medium at 40C. Rosettes were prepared immediately afterwards as described (Radaszkiewicz & Denk, 1975). CB was prepared as a stock solution in dimethylsulphoxide (DMSO) at a concentration of 5 mg CB in 0 5 ml DMSO. This stock solution was diluted with phosphate-buffered saline (PBS), pH 7 4, to double final CB-concentration and mixed with an equal amount of the lymphocyte suspension in RPMI 1640 medium (30 x 106 cells/ml). DMSO alone in identical dilution was used in control assays. In addition, for comparison the effect of NaCN in a concentration of 15 mm was tested on TBL and Th as described (Radaszkiewicz & Denk, 1975). To further evaluate the metabolic requirements for rosette formation and rosette stability additional experiments were performed in order to assess whether metabolic integrity of the lymphocyte is required to maintain an already formed rosette. For this purpose, NaCN (15 mm final concentration) was added to TBLRRBC rosette.
RESULTS CB proved to be a potent inhibitor of TBL-rosette formation (Table 1). Even in the low concentration of 2 pg/ml the rosette forming capacity was decreased to 5000 of the controls, 10 pug/ml showed already near maximal inhibition. In higher concentrations CB was as effective an inhibitor as NaCN. The solvent itself (DMSO) was ineffective. Th rosette formation was largely insensitive to CB in concentrations up to 20 pg/ml; at 40 ug/ml, however, some inhibition was observed (Table 1). Although NaCN in the concentration used (15 mM) inhibited TBL-rosette formation significantly as seen in Table 1, this drug was ineffective if added to the already formed rosette. For example, before addition of NaCN 21% rosette-forming TBL were found and after NaCN the value was at 23%4. TABLE 1. Effect of CB and NaCN on rosette formation by Th and TBL. The results of one representative experiment are listed (the experiments were repeated at least three times)
Percentage
rosette-forming cells*
Lymphocytes
NaCN
Cytochalasin B (pug/ml) 2
Tht TBLt
10
20
40
15 mM
76
80
78
37
78
(74)
(77)
(76)
(67)
(78)
9 (21)
7 (23)
6 (20)
5
5
(21)
(25)
* Numbers in parentheses are the percentages of the respective controls (DMSO in case of CB). t Pooled cells of four guinea-pigs.
DISCUSSION Metabolic activity is not an absolute requirement for spontaneous rosette formation between thymusrelated lymphocytes and RRBC in the guinea-pig model (Wilson & Coombs, 1973; Radaszkiewicz & Denk, 1975). Guinea-pig Th formed spontaneous rosettes in the live as well as in the dead state (Radaszkiewicz & Denk, 1974, 1975) whereas TBL did not if metabolically inhibited (Stadecker et al.,
Guinea-pig T cells-rabbit red cell rosettes
503
1973; Radaszkiewicz & Denk, 1975). Once established, however, TBL rosettes were not affected by metabolic inhibitors like cyanide. This indicates that metabolic cellular processes in the lymphocytes are only necessary to initiate but not to maintain the spontaneous rosette. CB, an inhibitor of microfilament function (Wessels et al., 1971) did not affect spontaneous rosette formation of Th but inhibited TBL rosettes. The different response of Th and TBL could be due to different receptor concentrations on the lymphocyte surface whereby a certain critical receptor density may be necessary for a stable rosette. In Th a high receptor density could support rosette formation even in the dead or metabolically inhibited state. In TBL with sparse receptors achievement of a critical density could require receptor movement and clustering upon contact with RRBC-a CBsensitive process. Clustering of surface receptors on various cells is well known (Ashman, 1973; Ashman & Raff, 1973; Singer, 1974; Thiele & Stark, 1974; Loor, Block & Little, 1975). Our experimental conditions make it unlikely that TBL rosette formation requires a typical capping of RRBC receptors since Yu (1974) has shown that capping of the erythrocyte receptors on human T cells counteracts rosette formation and is even the reason for the dissociation of an already formed rosette. Furthermore, as in B cells, classical capping of the erythrocyte receptor requires incubation at 370C (Yu, 1974) rather than the 4VC used in our experiments. The hypothesis of different receptor densities on Th and TBL is in line with the findings of several authors that certain thymus specific antigens are present in a higher concentration on the Th than on the TBL surface (Aoki et al., 1969; Raff, 1970). The higher amounts of contractile proteins in association with the TBL membrane (Fagraeus, The & Biberfeld, 1973; Fagraeus, Lidman & Biberfeld, 1974) would then appear as logical consequence to compensate, by increased active membrane mobility, for the lower receptor density. However, further evaluation of this hypothesis requires direct detection of the RRBC receptor on the lymphoid cell surface. This research was supported in part by Fonds zur Forderung der wissenschaftlichen Forschung (no. 1543) and by AdolfSonnleitner-Fonds of the Austrian Academy of Sciences.
REFERENCES AOKI, T., HAMMERLING, U., HARVEN, E. DE, BoYsE, E.A. & OLD, L.J. (1969) Antigenic structure of cell surfaces. An immunoferritin study of the occurrence and topography of H-2, 9, and TL alloantigens on mouse cells. I. exp. Med. 130, 979. ASHMAN, R.F. (1973) Lymphocyte receptor movement induced by sheep erythrocyte binding. 7. Immunol. 111, 212. ASHMAN, R.F. & RAFF, M. (1973) Direct demonstration of Theta-positive antigen-binding cells, with antigeninduced movement of thymus-dependent cell receptors. .7. exp. Med. 137, 69. BACH, J.F. (1973) Evaluation of T-cells and thymic serum factors in man using the rosette technique. Transplant. Rev. 16, 196. BAXLEY, G., BIsHoP, G.B., COOPER, A.G. & WORTIs, H.H. (1973) Rosetting of human red blood cells to thymocytes and thymus-derived cells. Clin. exp. Immunol. 15, 385. BRAIN, P., GORDON, J. & WILLETTS, W.A. (1970) Rosette formation by peripheral lymphocytes. Clin. exp. Immunol. 6, 681. COHNEN, G., FisCHER, K. & BRITTINGER, G. (1975) Human T-lymphocyte rosette formation. Inhibition by Cytochalasin B. Immunology, 29, 337. COOMBS, R.R.A., GuRr-ER, B.W., WILSON, A.B., HoLm, G. & LINDGREN, B. (1970) Rosette formation between human lymphocytes and sheep red cells not involving immunoglobulin receptors. Int. Arch. Allergy, 39, 658. FAGRAEUS, A., LIDMAN, K. & BIBERFELD, (G. (1974) Re-
action of human smooth muscle antibodies with human blood lymphocytes and lymphoid cell lines. Nature (Lond.), 252, 246. FAGRAEUS, A., THE, H. & BIBERFELD, G. (1973) Reaction of human smooth muscle antibody with thymus medullary cells. Nature: New Biology, 246, 113. FRoLAND, S.S. (1972) Binding of sheep erythrocytes to human lymphocytes. A probable marker of T-lymphocytes. Scand. J. Immunol. 1, 269. JoNDAL, M., HOLM, G. & WIGzELL, H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. 5. exp. Med. 136, 207. KERsEY, J.N., HOM, D.J. & BuTTRICK, P. (1974) Human T lymphocyte receptors for sheep erythrocytes: conditions for binding including inhibition by Cytochalasin B. 5. Immunol. 112, 862. LAY, W.H., MF.NDEs, N.F., BIANco, C. & NUSSENZWEIG, V. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531. LOOR, F., BLOCK, N. & LiTTLE, J.R. (1975) Dynamics of the TL antigens on thymus and leukemia cells. Cell. Immunol. 17, 351. AMNDEs, N.F., SARAIVA, P.J. & SANTOS, O.B.O. (1975) Restorative effect of normal human serum, transfer factor and thymosin on the ability of heated human lymphocytes to form rosettes with sheep erythrocytes. Cell. Immunol. 17, 560. RADASZKIEWICZ, T. & DENK, H. (1974) Identification of
504
T. Radaszkiewicz, D. Kotz & H. Denk
thymus dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes. Cell. Immunol. 14, 303. RADASZKIEWICZ, T. & DENK, H. (1975) Rosette formation of guinea pig lymphoid cells with rabbit erythrocytesdifferences between thymocytes and peripheral blood lymphocytes. Cell. Immunol. 16, 374. RAFF, M.C. (1970) Two distinct populations of peripheral lymphocytes in mice distinguishable by immunofluorescence. Immunology, 19, 637. SINGER, S.J. (1974) Molecular biology of cellular membranes with applications to immunology. Advances in Immunology (ed. by F. J. Dixon & H. G. Kunkel), volume 19, p. 1. Academic Press, New York. STADECKER, M.J., BISHOP, G. & WORTIS, H.H. (1973) Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells. J. Immunol. 111, 1834. THIELE, H.-G. & STARK, R. (1974) Common antibody-
induced redistribution on thymocytes of strain-speciesand non-species-specific antigenic determinants shared by brain and thymocytes of mice. Immunology, 27, 807. WESSELS, N.K., SPOONER, B.S., ASH, J.F., BRADLEY, M.O. LuDuENA, M.A., TAYLOR, E.L., WRENN, J.T. & YAMADA, K.M. (1971) Microfilaments in cellular and developmental process. Contractile microfilament machinery of many cell types is reversibly inhibited by cytochalasin B. Science, 171, 135. WILSON, A.B. & COOMBS, R.R.A. (1973) Rosette formation between guinea pig lymphoid cells and rabbit erythrocytes -a possible T-cell marker. Int. Arch. Allergy, 44, 544. WYBRAN, J., CARR, M.C. & FUDENBERG, H.H. (1972) The human rosette-forming cell as a marker of a population of thymus derived cells. J. cdin. Invest. 51, 2537. Yu, D.T.Y. (1974) Human lymphocyte receptor movement induced by sheep erythrocyte binding: Effect of temperature and neuraminidase treatment. Cell. Immunol. 14, 313.
