T cell mitogens and polyclonal B cell activators

[1]

T CELL MITOGENSAND PBA

3

[1] T Cell M i t o g e n s a n d P o l y c l o n a l B Cell A c t i v a t o r s

By GIOVANNI DI SABATO, JANICE M. HALL, and L U A N N THOMPSON

Introduction Lectins are divalent or polyvalent carbohydrate-binding proteins of plant or animal origin that are extensively used in biological research. Although a wide variety of biological effects has been ascribed to these substances, 1 lectins are primarily used for their agglutinating activity on erythrocytes and other cells and their mitogenic activity on lymphocytes (i.e., their ability to convert quiescent cells into growing and dividing "blasts"). The latter phenomenon often results in the production of various substances (lymphokines, immunoglobulins) involved in the regulation of the immune response. The binding activity of lectins is also used to separate various types of cells (e.g., tumor cells, 2 and thymocytes at various stages of maturation3). Affinity chromatography using lectin-linked gels is a valuable technique for the separation of cells or carbohydrate-containing substances (see this volume [37]). Elution is usually carded out with carbohydrate having specific affinity for the lectin used. This chapter deals primarily with the method used to study lectins as mitogens for lymphoid cells. It should be pointed out, however, that not all lectins are mitogenic and not all mitogens are lectins. Lectins bind to receptors situated on the lymphocyte surface. Binding, however, does not necessarily mean mitogenicity. A number of lectins (e.g., wheat germ agglutinin and the agglutinin of Dolichos biflorus) bind but are not mitogenic. General reviews on various aspects of the biology and biochemistry of lectins have recently appeared. 1'3-7 Other publications S. H. Baronides, Annu. Rev. Biochem. 50, 207 (1981). 2 N. Sharon and H. Lis, Science 177, 949 (1972). 3 y . Reisner and N. Sharon, this series, Vol. 108 [17]. 4 N. Sharon, Adv. Immunol. 34, 213 (1983). 5 D. A. Hume and M. J. Weidemann, "Mitogenic Lymphocyte Transformation." Elsevier/ North-Holland, Amsterdam, 1981. 6 K. Resch and H. Kirchner, eds., "Mechanisms of Lymphocyte Activation." Elsevier/ North-Holland, Amsterdam, 1982. 7 M. W. Elves, "The Lymphocyte." Year Book Med. Publ., Chicago, Illinois, 1972.

METHODS IN ENZYMOLOGY, VOL. 150

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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STIMULATION OF LYMPHOCYTES

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have dealt with the study of the morphological and biochemical 8 changes taking place in transformed lymphocytes. Assay of Mitogenic Activity

Materials 96-Well Linbro microplates (U shaped), sterile with lids (Flow Labs) Constriction pipets (Fisher) of various sizes or automatic pipets in the 5- to 1000-/zl range RPMI-1640 medium or equivalent Appropriate, heat-inactivated (45 min at 56°) serum (human, fetal calf, horse) Stock solution of the mitogen at the appropriate concentration (100-500 /zg/ml)

Procedure Prepare a 10% solution of serum in RPMI containing 5-20/zg/ml of the mitogen. Distribute 200/zl in each well of the microplate (each sample is usually run in triplicate). Add 25/zl of target cells (e.g., spleen, lymph node lymphocytes, or thymocytes) at a density of 4-20 x 106 ml. The number of cells in each well will be 0.1-0.5 x 106. The places are incubated for 24-48 hr at 37 ° in a humidified atmosphere of 95% air-5% CO2. Each culture is then labeled by adding with a repetitive syringe (Hamilton Co., Reno, NV) 10/zl (1/zCi) of [methyl-3H]thymidine (ICN, specific activity 60-90 Ci/nmol). The cultures are incubated for an additional 24 hr under the same conditions, harvested with an automatic harvester, and the radioactivity is measured in a scintillation counter. Parameters Affecting Lymphocyte Transformation The amount of radioactivity incorporated in the cells (indicating the degree of mitogenicity of a lectin) varies greatly in function of a number of parameters. Serum. Fetal calf serum or adult calf serum is often used to study the mitogen-induced transformation of cells. We routinely use pooled human serum which is as satisfactory as fetal or adult calf serum and has the added advantage that it makes bacterial contamination less likely. The serum should be kept frozen in aliquots of 2-5 ml and defrosted just before use. Repeated freezing and thawing should be avoided. Using 8 T h i s v o l u m e [3].

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T CELL MITOGENSAND PBA

5

phytohemagglutinin (PHA) as the mitogen we have observed only minor differences in the amount of labeled thymidine incorporated into murine spleen lymphocytes with serum concentration ranging from 5 to 20%. With concanavalin A (Con A), however, the concentration of serum has a great effect on the mitogenic response of lymphoid cells, due to the binding of Con A to serum proteins that makes less mitogen available to stimulate the c e l l s . 9 Mitogen. The dose-response curve for most mitogens is bell shaped. In other words, maximum stimulation takes place at a certain mitogen concentration below and above which stimulation is less or fails to take place in spite of binding of the mitogen to the target cells. The amount of mitogen that produces an optimal response in cells varies greatly with the mitogen and with the type of target cell. For instance, in our hands, 0.3 x 106 murine AKR/J spleen cells per well, 10% pooled human serum, a 40-hr incubation (30 before and 10 after labeling with thymidine), and 0.4/zg of PHA (Burroughs Wellcome) result in the incorporation of 40-60 x 103 cpm/culture~ The efficiency of the scintillation counter is 40-60%. Blank cultures (in the absence of mitogen) usually incorporate 200-1000 cpm. The best way to establish the optimal dose of mitogen consists of making log2 dilutions in the wells of the microplate starting with an initial concentration of the mitogen of 1-2 /zg/well. Among the suppliers of lectins are: E-Y Laboratories, Inc., San Mateo, California, and Pharmacia, Inc., Piscataway, New Jersey. • Target Cells. Most mitogenic lectins act on T lymphocytes. Some are mitogenic for B lymphocytes (see below). Human peripheral blood lymphocytes (prepared according to the technique described in this series, Vol. 108 [9]) and murine spleen, lymph node, or thymus cells (this series, Vol. 108 [6]) are commonly used in this type of study. Stimulation is usually polyclonal, in the sense that a relatively large proportion (30-60%) of the target cells is stimulated. It should be pointed out, however, that PHA has little mitogenic effect on murine thymocytes since it stimulates only mature T cells, comprising just 5-10% of the total thymus cell population. Con A (another commonly used T cell mitogen) stimulates equally well mature and immature T lymphocytes and is therefore mitogenic for all thymocytes. Radioactive Label. [methyl-3H]Thymidine is usually purchased as an ethanol:water (7:3) solution (specific activity 60-90 Ci/mmol) and is diluted in phosphate-buffered saline to contain 1 /xCi/10 /zl. In our experience, this diluted solution of [methyl-3H]thymidine can be kept at least 1 year in a frozen state without appreciable decomposition. The 9 D. M. Chen and G. DiSabato, l m m u n o l . C o m m u n . 6, 395 (1977). (text continues on p. 15)

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STIMULATION OF LYMPHOCYTES

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TABLE I LECTINS FROM PLANT AND ANIMAL SOURCES References"

Lectin

Aaptos papillata Abramis brama Abrus precatorius Actinomyces viscosus Adenia digitata Agariues bisporus Agaricus campestris Agaricus edulis Aleuria aurantia Allomyrina dichotoma Amphicarpea bracteata Amphitrite ornata Androctomus australis Anguilla anguilla Anguilla rostrata Anthocidaris crassispina Aplysia californica Aplysia dactylomela Aplysia depibans Aplysia juliana Aplysia kurodai Arachis hypogea Arianta arbustorum Arion empiricorum Artocarpus integrifolia Asteria forbesi Axinella polypoides Bahuinia purpurea Bahuinia variegata Bandeiraea simplicifolia Biomphalaria glabrata Birgus latro Botrylloides leachii Botrox atrox Brachypodium sylvaticum Bradybaena fruticum Bryonia dioica Butea frondosa Callinectes sapidus Canavalia ensiformis Cancer antennarius Carabia sativa Caragana arborescens

Purification and characterization

Carbohydrate specificity

1, 2, 5

1, 5

3

3

4-6

5, 7

9

9

10-12 5, 13 5, 14 17 18 20

11 5, 13 5, 15 17 18 20

-2!

Mitogenicity

13 16 19

-21

2 5, 22, 23 24 26

5, 22 24, 25 26

22

2

5, 28

27 27 27 27 5, 28

2

2

2, 29 30 2, 32 2, 33 5, 35, 36

29

27

5, 39, 40 42 2, 44, 45 47

27

28

31 34 5, 35 38 5, 39-41 42 43 44, 45 46 47

2

2

48 49 50

48 49 50, 51

34 37

5

5

52, 53

54 55 5, 56

54 55 5, 56

56

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T CELL MITOGENS AND P B A

7

TABLE I (continued) References ~

Lectin

Carcinoscorpius rotunda cauda Cepaea memoralis Cerastium tormentosum Cherax destructor Cicer arietinum Clitocybe nebularis Conavalia ensiformis Conavalia ensiformis Coregonus lavaretus maraena Coronilla varia Crassostrea gigas Crassostrea virginica Crotalaria juncea Crotolaria zanzibarica Croton tiglium Cryptoderma citrinum Cucumis melo Cucumis sativus Cucurbita maxima Cucurbita pepo Cyathiopodia macropus Cytisus scoparius Cytisus sessifolius Datura stramonium Dextran sulfate Dictyostelium discoideum Dictyostelium purpureum Didemnum candidum Dioclea grandiflora Discina perlata Dolichos biflorus Dolichos lablab Electrophorus electricus Erythrina arborescens Erythrina corallodendrum Erythrina cristagalli Erythrina indica Erythrina lithosperma Erythrina suberosa Erythrina variegata Euhadra callizona amaliae Euphorbia characias Euonimus europaeus Euphorbia heterophylla

Purification and characterization

Carbohydrate specificity

57-59 60 61 2 62 22 5 63-66 67 38 68 2, 69, 70 5, 71 38 72

57-59 60 61 2 62 22 5 63-66 67 38 68 2, 69, 70 5, 71 38 72

73 73 74, 75 73

73 73 74, 75 73

76 5, 77, 79 80-82

76 5, 78 80-83

5, 85 86 87 89, 90

5, 85 86 88 89, 90

5, 60 93 5, 94 95 96 97 49 95 95 99 2 100 5, 101, 102, 104 105

5, 91 93 5, 94 95 96 98 95 95 95 99 2 100 5, 101-103 105

Mitogenicity

60

62 22 5 63-66 67 68

19

19

80 84

19 91, 92

96 97

100 103 (continued)

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STIMULATION OF LYMPHOCYTES

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TABLE I (continued) References"

Lectin

Flammulina veltipes Fomes fomentarius Fomitopsis cytisina Geodia cydonium Glycera dibranchiata Glycine max Griffonia simplicifolla (see Bandeiraea simplicifolia) Grifola frondosa Grifola umbellata Halocynthia hilgendorfi Halocynthia pyriformis Halocynthia roretzi Helix aspersa Helix hortensis (see Cepaea nemoralis) Helix lucorum Helixpomatia Hemicentrotus pulcherrimus Holothuria polii Holothuria tubulosa Homerus americanus Hononis hircina Hordeum vulgare Hura crepitans Laburnum alpinum Lactuca scariole Lathyrus ochrus Lathyrus odoratus Lathyrus sativus Lens culinaris Lens esculenta (see Lens culinaris)

Purification and characterization

Carbohydrate specificity

106 22

106 22

107 108 5, 109

107 108 5, 110

Mitogenicity 106 22 19 107 111

19 19 112 2, 87 114 2

113 114 2, 115

60 2, 5, 22

60 2, 5, 22

2 2 2

2 2 2

2, 5, 118, 119 121 122 100, 123

2, 5, 118, !19 121 122 100, 123

5

5

124a 125 126 128-130 5, 131-133

124a 125 127 129 5, 131-133

126 129 133, 134

135 2, 5, 136 5 108, 139 140-144 5, 145 5, 22, 146, 147 22

135 2, 5, 136 5 139 140, 142-144 5, 145 5, 22 22

137 138 108 140 145 22 22

2

2

116, 117

120 121 123, 124 124a

Lima bean (see Phaseolus lunatus)

Limax flavus Limulus poliphemus Lotus tetragonolobus Lumbricus terrestris Lycopersicon esculentum Maackia amurensis Maclura pomifera Marasmius oreades Mercenaria mercenaria

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T CELL MITOGENS AND P B A

9

TABLE I (continued) References"

Lectin

Molchella esculenta Momordia charantia Octopus vulgaris Onobrychis viciifolia Ononis hirchina Ononis spinosa Oryza sativa Otala lactea Panuliris argus Panulirus interruptus Pelargonium hortorum Perca fluviatilis Petromyzon marinas Peziza vesciculosa Phaseolus aureus (see Vigna radiata) Phaseolus coccineus Phaseolus limensis (see Phaseolus lunatus) Phaseolus lunatus Phaseolus vulgaris Phytolacca americana Phytolacca esculenta Pieris brassicae Pisam sativum Pokeweed mitogen (see Phytolacca americana) Pollen mitogens Polyporus fumosum Polyporus rugulosus Polyporus vinosus Polysphondylium pallidum Procambarus clarkii Pseudocentrotus depressus Pseudomonas aeruginosa Psophocarpus tetragonolobus Rhodnius prolixus Ricinus communis Robinia pseudoacacia Rumex crispus Rutilus rutilus Sambucus nigra Sarcophaga peregrina Sarothamnus scoparius

Purification and characterization

Carbohydrate specificity

49, 148 2 149, 150 121 22 152 153 2, 154 155 156 3 157 19

49, 148 2 14%151 121 22 152 153 154 155 156 3

158

158

158

5, 159, 160 5, 162 5, 165 5, 166 167 5, 168, 169

5, 159, 160 5, 163 5, 165 5 167 5, 168

160, 161 164 165 166

169a

169a

Mitogenicity 19

121 22 152

156

19

168

19 19 19 5, 169b 2 2 96, 170, 171 172, 173 174 5, 175 5, 124, 176 156 3 178 179, 180 22, 181

5, 169b 2 2 96, 170, 171 172, 173 174 5, 8 5, 124 156 3 178 179 181

96, 170

8 176, 177 156

181

(continued)

l0

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STIMULATION OF LYMPHOCYTES TABLE l (continued) References"

Lectin Saxidomus giganteus Saxidomus purpuratus Scardinus erythrophtalmus Secale cereale Sesanum indicum Slime mold Snake venom Solanum tuberosum Sophorajaponica Stereum membranaceum Styela plicata Tachpleus tridentatus Trichosanthes kirilowii Tridacna derasa Tridacna gigas Tridacna maxima Triticum vulgaris Ulex europeus Ulex gaUi Ulex nanus Ulex parviflorus Velesunio ambiguus Vicia cracca Vicia cretica Vicia ervilia Viciafaba Vicia graminea Vicia sativa Vicia villosa Vigna radiata Vimba vimba Viscum album Volnariella volvacea Wistaria floribunda Xenopus laevis

Purification and characterization

Carbohydrate specificity

i 82 183 3 122 184 185 186 5, 82, 187-189 5, 190

182 183 3 122 184 185 186 5, 78, 187-189 5, 190

Mitogenicity

-19

87 191, 192 193 194 194 5, 195 5, 196 5,200-202 38 38 38 2, 203 5, 22, 204, 205 206 5, 207 5, 208-211 5, 212, 213 214, 215 216, 220 221 3 222 223 5, 224-226 228, 229

87 191, 192 193 194 194 5, 195 5, 197 5, 200 38 38 38 203 5, 22, 204, 205 206 5, 207 5, 211 5,212, 213 214, 215 216, 217, 220 221 3 222 223 5, 224 228, 229

198, 199 202

22

214, 215 218, 219

222 224, 225,227

° Key to references: (1) H. Bretting, E. A. Kabat, J. Liao, and M. E. A. Pereira, Biochemistry 15, 5029 (1976); (2) P. L. Ey, and C. R. Jenkins, in "The Reticuloendothelial System: A Comprehensive Treatise" (N. Cohen and M. M. Sigel, eds.), Vol. 3, p. 321. Plenum, New York, 1982; (3) A. Krajhanski, V. Horejsi, and J. Kocourek, Biochim. Biophys. Acta 532, 215 (1978); (4) M. S. Herrmann and W. D. Behnke, Biochim. Biophys. Acta 667, 397 (1981); (5) I. J. Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978); (6) S. Olsnes, E. Saltvedt, and A. Pihl, J. Biol. Chem. 249, 803 (1974); (7) S. J. Kaufman and A. McPherson, Cell (Cambridge, Mass.) 4, 263 (1975); (8) O. Closs, E. Salvedt, and S. Olsnes, J. lmmunol. 115, 1045 (1975); (9) J. O. Cisar, E. L. Barsumian, S. H. Curl, A. E. Watter, A. L. Sandberg, and

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T CELL MITOGENS AND P B A

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References to TABLE I (continued) R. P. Siraganian, J. lmmunol. 127, 1318 (1981); (10) L. Barbieri, M. Zamboni, L. Montanaro, S. Sperti, and F. Stirpe, Biochem. J. 185, 203 (1980); (I1) S. OIsnes, T. Haylett, and K. Refsnes, J. Biol. Chem. 253, 5069 (1978); (12) A. Gasperi-Campani, L. Barbieri, E. Lorenzoni, L. Montanaro, S. Sperti, E. Bonetti, and F. Stirpe, Biochem. J. 174, 491 (1978); (13) G. A. Presant and S. Kornfeld, J. Biol. Chem. 247, 6937 (1972); (14) H. J. Sage and S. L. Connett, J. Biol. Chem. 244, 44713 (1969); (15) H. J. Sage and J. J. Vasquez, J. Biol. Chem. 242, 120 (1967); (16) N. M. Young, M. A. Leon, T. Takahashi, I. K. Howard, and H. J. Sage, J. Biol. Chem. 246, 1596 (1971); (17) R. Eifler and P. Ziska, Experientia 36, 1285 (1980); (18) N. Kochibe and K. Furukawa, Biochemistry 19, 2841 (1980); (19) T. Yadomae, I. Suzuki, H. Yonekubo, K. Nunomura, and T. Miyazaki, Microbiol. lmmunol. 23, 815 (1979); (20) K. Umetsu, S. Kosaka, and T. Suzuki, J. Biochem. (Tokyo) 95, 239 (1984); (21) S. J. Garte and C. S. Russell, Biochim. Biophys. Acta 439, 368 (1976); (22) V. Horejsi and J. Kocourek, Biochim. Biophys. Acta 538, 299 (1978); (23) C. Kelly, Biochem. J. 220, 221 (1984); (24) G. F. Springer, T. Takahashi, P. R. Desai, and B. J. Kolecki, Biochemistry 4, 2099 (1965); (25) G. F. Springer and P. R. Desai, Biochemistry 10, 3749 (1971); (26) H. Sasaki and K. Aketa, Exp. Cell Res. 135, 15 (1981); (27) N. Gilboa-Garber, A. J. Susswein, L. Mizrahi, and D. Avichezer, FEBS Lett. 181, 267 (1985); (28) R. Lotan and N. Sharon, this series, Vol. 50 [41]; (29) L. Habets, U. C. Vieth, and G. Hermann, Biochim. Biophys. Acta 582, 154 (1979); (30) M. C. Roque-Barreira and A. CamposNeto, J. lmmunol. 134, 1740 (1985); (31) M. M. Bunn-Moreno and A. Campos-Neto, J. lmmunol. 127, 427 (1981); (32) C. L. Finstad, G. W. Litman, J. Finstad, and R. A. Good, J. lmmunol. 108, 1704 (1972); (33) H. Bretting and E. A. Kabat, Biochemistry 15, 3228 (1976); (34) S. G. Phillips, H. Bretting, and E. A. Kabat, J. Immunol. 117, 1226 (1976); (35) T. Osawa, T. Irimura, and T. Kawaguchi, this series, Vol. 50 [42]; (36) A. Hishimuna, Y. Imai, T. Nakano, and T. Osawa, Int. Arch. Allergy Appl. Immunol. 72, 330 (1983); (37) T. Kawaguchi and T. Osawa, Biochemistry 15, 4581 (1976); (38) T. Kristiansen, this series, Vol. 34 [31]; (39) L. A. Murphy and I. J. Goldstein, this series, Vol. 50 [38] and [39]; (40) F. M. Delmotte and I. J. Goldstein, Eur. J. Biochem. 112, 219 (1980); (41) C. Wood, E. A. Kabat, L. E. Murphy, and I. J. Goldstein, Arch. Biochem. Biophys. 198, 1 (1979); (42) H. Bretting, E. Stanislawski, G. Jacobs, and W. Becker, Biochim. Biophys. Acta 749, 143 (1983); (43) J. E. Cushing, Fed. Proc., Fed. Am. Soc. Exp. Biol. 26, 1666 (1967); (44) S. F. Schluter, P. L. Ey, D. R. Keough, and C. R. Jenkin, Immunology42, 241 (1981); (45) D. R. Coombie, P. L. Ey, S. F. Schluter, and C. R. Jenkin, Immunology 42, 661 (1981); (46) S. Khalap, C. F. Phelps, T. E. Thompson, and E. R. Gold, Vox Sang. 22, 89 (1972); (47) W. Peumans, C. Spaepen, H. M. Stinissen, and A. R. Cadier, Biochem. J. 205, 635 (1982); (48) W. J. Peumans, M. Nsimba-Lubaki, A. R. Carlier, and E. van Driessche, Planta 160, 222 (1984); (49) V. Horjsi, M. Ticha, J. Novotny, and J. Kocourek, Biochim. Biophys. Acta 623, 439 (1980); (50) G. B. Pauley, Contemp. Top. lmmunobiol. 4, 241 0975); (51) G. B. Pauley, Experientia 29, 210 (1973); (52) A. E. Powell and M. A. Leon, Exp. Cell Res. 62, 315 (1970); (53) D. M. Chen and G. Di Sabato, lmmunol. Commun. 6, 395 (1977); (54) M. H. Ravindranath, H. H. Higa, E. L. Cooper, and J. C. Paulson, J. Biol. Chem. 260, 8850 0985); (55) C. S. Tumosa, Experientia 49, 718 (1984); (56) R. Bloch, J. Jenkins, J. Roth, and M. M. Burger, J. Biol. Chem. 251, 5929 (1976); (57) S. Bishayee and D. T. Dorai, Biochim. Biophys. Acta 623, 89 (1980); (58) D. T. Dorai, S. Spiral, S. Mohan, B. K. Bachhawat, and T. S. Balganesh, Biochem. Biophys. Res. Commun. 104, 141 0982); (59) D. T. Dotal, B. K. Bachhawat, S. Bishayee, K. Kannan, and D. R. Rao, Arch. Biochem. Biophys. 209, 325 (1981); (60) P. D. Zalewski, I. J. Forbes, G. Uhlenbruck, and L. Valente, Clin. Exp. lmmunol. 44, 304 (1981); (61) G: W. G. Bird (continued)

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STIMULATION OF LYMPHOCYTES

[1]

References to TABLE I (continued) and J. Wingham, Vox Sang. 19, 132 (1970); (62) J. Kolberg, T. E. Michaelsen, and K. Sletten, Hoppe-Seyler's Z. Physiol. Chem. 364, 655 (1983); (63) A. Novogrodski and E. Katchalski, Biochim. Biophys. Acta 228, 579 (1971); (64) J. L. Wang and G. M. Edelman, J. Biol. Chem. 253, 3000 (1978); (65) M. Beppu, T. Tadao, and T. Osawa, J. Biochem. (Tokyo) 85, 1275 (1979); (66) T. Osawa, this volume [2]; (67) A. Krajhanzi, V. Horejsi, and J. Kocourek, Biochim. Biophys. Acta 532, 209 (1978); (68) S. W. Hardy, A. W. Thomson, and T. C. Fletcher, Comp. Biochem. Physiol. A 60A, 473 (1978); (69) R. T. Acton, J. C. Bennett, E. E. Evans, and R. E. Schronenloher, J. Biol. Chem. 244, 4128 (1969); (70) G. R. Vasta, T. C. Cheng, and J. J. Marchalonis, Cell. lmmunol. 88, 475 (1984); (71) B. Ersson, K. Aspberg, and J. Porhth, Biochim. Biophys. Acta 310, 446 (1973); (72) K. K. Banerjee and A. Sen, Arch. Biochem. Biophys. 212, 740 (1981); (73) A. K. Allen, Biochem. J. 183, 133 (1979); (74) C. Weber, W. W. Franke, and J. Kartenbeck, Exp. CellRes. 87, 79 (1974); (75) D. D. Sabnis and J. W. Hart, Planta 142, 97 (1978); (76) M. M. Young, D. C. Watson, and R. E. Williams, Arch. Biochem. Biophys. 222, 41 (1984); (77) I. Matsumoto and T. Osawa, Biochemistry 13, 582 (1974); (78) I. Matsumoto and T. Osawa, Vox Sang. 21, 548 (1971); (79) T. Osawa, Biochim. Biophys. Acta 115, 507 (1966); (80) J. F. Crowley and I. J. Goldstein, this series, Vol. 83 [30]; (81) N. N. Desai, A. K. Allen, and A. Neuberger, Biochem. J. 197, 345 (1981); (82) D. Ashford, N. N. Desai, A. K. Allen, A. Neuberger, M. A. O'Neill, and R. R. Selvendran, Biochem. J. 2,111, 199 (1982); (83) J. F. Crowley, I. J. Goldstein, J. Arnarp, and J. Lonngren, Arch. Biochem. Biophys. 231, 524 (1984); (84) R. Palacios, I. Sugawara, and C. Fernandez, J. lmmunol. 128, 621 (1982); (85) S. H. Barondes, S. D. Rosen, W. A. Frazier, D. L. Simpson, and P. L. Haywood, this series, Vol. 50 [31]; (86) D. N. Cooper and S. H. Barondes, J. Biol. Chem. 256, 5046 (1981); (87) G. R. Vasta and J. J. Marchalonis, Prog. Clin. Biol. Res. 157, 125 (1984); (88) G. R. Vasta, G. W. Warr, and J. J. Marchalonis, Comp. Biochem. Physiol. B 73B, 887 (1982); (89) R. A. Noreira, A. C. H. Barros, J. C. Stewart, and A. Pusztai, Planta 158, 63 (1983); (90) M. Richardson, F. D. A. P. Campos, R. A. Moreira, I. L. Alnovz, R. Regrie, W. B. Watt, and A. Pusztai, Eur. J. Biochem. 144, 101 (1984); (91) W. G. Carter and M. E. Etzler, J. Biol. Chem. 250, 2756 (1975); (92) H. J. Downing, G. C. M. Kemp, and M. A. Denborough, Nature (London) 217, 654 (1968); (93) A. Guran, M. Ticha, K. Filka, and J. Kokourek, Biochem. J. 209, 653 (1983); (94) G. Levi and V. I. Teichberg, J. Biol. Chem. 256, 5735 (1981); (95) C. Battacharyya, P. K. Das, and A. Sen, Arch. Biochem. Biophys. 211, 459 (1981); (96) N. Gilboa-Garber and L. Mizrahi, Can. J. Biochem. 59, 315 (1981); (97) J. L. Iglesias, H. Lis, and N. Sharon, Eur. J. Biochem. 123,247 (1982); (98) P. M. Kaladas, E. A. Kabat, J. L. Iglesias, H. Lis, and N. Sharon, Arch. Biochem. Biophys. 217, 624 (1982); (99) T. K. Datta and P. S. Basu, Biochem. J. 197, 751 (1981); (100) L. Barbier, A. Falasca, C. Franceschi, F. Licastro, C. A. Rossi, and F. Stirpe, Biochem. J. 215, 433 (1983); (101) F. Pacak and J. Kocourek, Biochim. Biophys. Acta 400, 374 (1975); (102) J. Petryniak, M. E. A. Pereira, and E. A. Kabat, Arch. Biochem. Biophys. 178, 118 (1977); (103) J. Petryniak, D. Dus, and J. Podwinska, Eur. J. lmmunol. 13, 459 (1983); (104) D. R. Schultz and P. I. Arnold, Mol. lmmunol. 19, 1681 (1982); (105) N. Nsimba-Lubaki, W. J. Peumans, and A. R. Carlier, Biochem. J. 215, 141 (1983); (106) M. Isuda, J. Biochem. (Tokyo) 86, 1463 (1979); (107) H. Bretting, S. G. Phillips, H. J. Klumpart, and E. A. Kabat, J. lmmunol. 127, 1652 (1981); (108) E. L. Cooper, E. A. Stein, and A. Wodjani, Prog. Clin. Biol. Res. 157, 43 (1984); (109) J. A. Gordon, S. Blumberg, H. Lis, and N. Sharon, FEBS Lett. 24, 193 (1972); (110) H. Lis, B.-A. Sela, L. Sachs, and N. Sharon, Biochim. Biophys. Acta 211, 582 (1970); (111) A. Novogrodski and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A. 70, 2515 (1973); (112) M. T. Fuke and T. Sugai, Biol. Bull. (Woods Hole, Mass.) 143, 140 (1972); (113) R. S. Anderson and R. A. Goad, Biol. Bull. (Woods Hole, Mass.) 148, 357 (1975); (114) Y.

[1]

T CELL MITOGENS AND P B A

13

References to TABLE I (continued) Yokosawa, H. Sawada, Y. Abe, T. Numakunai, and S. Ishii, Biochem. Biophys. Res. Commun. 107, 451 (1982); (115) M. Bizot, Vox Sang. 21, 465 (1971); (116) M. Wiken, U. HeUstr6m, and P. Perlmann, Eur. J. Immunol. 14, 1003 (1984); (117) B. Axelssow, A. Kimura, S. Hammarstr6m, H. Wigzell, K. Nilsson, and H. Melstedt, Fur. J. lmmunol. 8, 757 (1978); (118) J. L. Hall and D. T. Rowlands, Jr., Biochemistry 13, 821 (1974); (119) J. L. Hall and D. T. Rowlands, Jr., Biochemistry 13, 828 (1974); (120) L. R. Herron, C. A. Abel, J. Vanderwai1, and P. A. Campbell, Eur. J. lmmunol. 13, 73 (1983); (121) V. Horejsi, O. Cheloupecka, and J. Kocourek, Biochim. Biophys. Acta 539, 287 (1978); (122) W. J. Peumans, H. M. Stuinissen, and A. R. Carlier, Biochem. J. 203, 239 (1982); (123) A. Faiasca, C. Franceschi, C. A. Rossi, and F. Stirpe, Biochim. Biophys. Acta 632, 95 (1980); (124) A. McPherson and S. Hoover, Biochem. Biophys. Res. Commun. 89, 713 (1979); (124a) S. E. Yen, J. M. Mansfield, and J. H. Wallace, Int. Arch. Allergy Appl. lmmunol. 61, 32 (1980); (124b) S. E. Yen, J. M. Mansfield, R. F. Levine, and J. H. Wallace, Int. Arch. Allergy Appl. Immunol. 65, 445 ( 1981); (125) M. Richardson, P. Rouge, B. Sousa-Cavada, and A. Yarwood, FEBS Lett. 175, 76 (1984); (126) J. Kolberg, Acta Pathol. Microbiol. Scand., Sect. C C86, 99 (1978); (127) J. Kolberg and T. E. Michaelsen, Acta Pathol. Microbiol. Scand., Sect. C C87, 275 (1979); (128) K. Sletten and J. Kolberg, Hoppe-Seyler's Z. Physiol. Chem. 364, 1047 (1983); (129) J. Koiberg and K. Sletten, Biochim. Biophys. Acta 704, 26 (1982); (130) B. K. Dutta, R. Chatterjee-Ghose, and A. Sen, Arch. Biochem. Biophys. 201, 137 (1980); (131) I. K. Howard and H. J. Jage, Biochemistry 8, 2436 (1969); (132) S. Toyoshima, T. Osawa, and A. Tonomura, Biochim. Biophys. Acta 221, 514 (1970); (133) M. Ticha, G. Entlicher, J. V. Kostir, and J. Kochourek, Biochim. Biophys. Acta 221, 282 (1970); (134) A. Hishimuna, Y. Imai, T. Nakano, and T. Osawa, Int. Arch. Allergy Appl. Immunol. 72, 330 (1983); (135) R. L. Miller, J. F. Collawn, Jr., and W. W. Fish, J. Biol. Chem. 257, 7574 (1982); (•36) S. H. Barondes and T. P. Nowak, this series, Vol. 50 [30]; (137) A. C. Roche, Y. Perrodon, B. Halpern, and M. Monsigny, Eur. J. Immunol. 7, 263 (1977); (138) R. L. P. Flower and G. E. Wilcox, J. Immunol. Methods 46, 347 (1981); (139) E. A. Stein and E. L. Cooper, Comp. Biochem. Physiol. B 76B, 197 (1983); (140) M. S. Nachbar and J. D. Oppenheim, this series, Vol. 83 [29]; (141) D. C. Kilpatrick, Biochem. J. 185, 269 (1980); (142) M. S. Nachbar, J. D. Oppenheim, and J. O. Thomas, J. Biol. Chem. 255, 2056 (1980); (143) D. C. Kilpatrick, J. Weston, and S. J. Urbaniak, Anal. Biochem. 134, 205 (1983); (144) D. C. Kilpatrick, C. Graham, S. J. Urbaniak, C. E. Jeffree, and A. K. Allen, Biochem. J. 220, 843 (1984); (145) T. Kawaguchi, I. Matsumoto, and T. Osawa, J. Biol. Chem. 249, 2786 (1974); (•46) J. N. Bausch, J. Rickey, and R. D. Por6tz, Biochemistry 20, 2618 (1981); (147) M. Sarkar, A. M. Wu, and E. A. Kabat, Arch. Biochem. Biophys. 209, 204 (1981); (148) T. Mazumider, N. Gaur, and A. Surolia, Eur. J. Biochem. 113, 463 (1981); (149) N. M. Young, R. E. Williams, C. Roy, and M. Yaguchi, Can. J. Biochem. 60, 933 (1982); (150) K. D. Hapner and J. E. Robbins, Biochim. Biophys. Acta 580, 186 (1979); (151) A. E. Namen and K. D. Hapner, Biochim. Biophys. Acta 580, 198 (1979); (152) M. J. Isuda, Biochemistry 86, 145 (1979); (153) S. Matsubara and W. C. Boyd, Science 183, 339 (1974); (154) R. T. Acton, P. F. Weinheimer, and W. Niedermeier, Comp, Biochem. Physiol. B 44B, 185 (1973); (155) A. Tyler and C. Netz, J. Exp. Zool. 100, 387 (1945); (156) H. J. Downing, G. C. M. Kemp, and N. A. Denborough, Nature (London) 217, 654 (1968); (157) J. J. Marchalonis and G. M. Edelman, J. Exp. Med. 127, 891 (1968); (158) P. Angelisova and C. Hasklovec, Eur. J. Biochem. 83, 163 (1978); (159) E. R. Pandolfino and J. A. Magnuson, J. Biol. Chem. 255, 870 (1980); (•60) E. R. Pandolfino, A. E. Namen, G. R. Munske, and J. A. Magnuson, J. Biol. Chem. 258, 9203 (1983); (161) G. R. Munske, E. R. Pandolfino, and J. A. Magnuson, J. lmmunol. 127, 1607 (1981); (162) D. A. Rigas and E. E. Osgood, J. Biol. Chem. 212, 607 (1955); (continued)

14

STIMULATION OF LYMPHOCYTES

[1]

References to TABLE I (continued) (163) R. Kornfeld and S. Kornfeld, J. Biol. Chem. 245, 2536 (1970); (164) T. H. Weber, H. Aro, and C. T. Nordman, Biochim. Biophys. Acta 263, 94 (1972); (165) M. J. Waxdel, Biochemistry 13, 3671 (1974); (166) H. Tokuyama, Biochim. Biophys. Acta 317, 338 (1973); (167) B. Mauchamp, Biochimie 64, 1001 (1982); (168) I. S. Trowbridge, Proc. Natl. Acad. Sci. U.S.A. 70, 3650 (1973); (169) C. Richardson, W. D. Behnke, J. H. Freischeim, and K. M. Blumenthal, Biochim. Biophys. Acta 537, 310 (1978); (169a) F. J. Anfosso, P. M. Guillard, and J. P. Charpin, Int. Arch. Allergy Appl. Immunol. 71, 6 (1983); (169b) M. J. Waxdal, this series, Vol. 50 [40]; (170) N. Gilboa-Garber, Biochim. Biophys. Acta 273, 165 (1972); (171) N. Gilboa-Garber, L. Mizrahi, and N. Garber, FEBS Lett. 28, 93 (1972); (172) S. G. Pueppke, Biochim. Biophys. Acta 581, 63 (1979); (173) A. A. Kortt, Arch. Biochem. Biophys. 236, 544 (1985); (174) M. E. A. Pereira, A. F. B. Andrade, and J. M. C. Ribeiro, Science 211, 597 (1981); (175) T. T.-S. 'Lin and S.-L. Li, Eur. J. Biochem. 105, 453 (1980); (176) A. Sharif and R. Bourrillon, Cell. lmmunol. 19, 372 (1975); (177) A. Shafif, J. Brochier, and R. Bourrillon, Cell. lmmunol. 31, 302 (1977); (178) W. F. Broekaert, M. Nsimba-Lubaki, B. Peeters, and W. J. Peumans, Biochem. J. 221, 163 (1984); (179) H. Komano, D. Mizuno, and S. Natori, J. Biol. Chem. 255, 2919 (1980); (180) H. Takahashi, H. Komano, N. Kawaguchi, N. Kitamura, S. Nakanishi, and S. Natori, J. Biol. Chem. 260, 12228 (1985); (181) L. G. Gurtler, Biochim. Biophys. Acta 544, 593 (1978); (182) H. M. Johnson, Science 146, 548 (1964); (183) M. Tatsumi, Y. ArM, and T. ltoh, J. Biochem. (Tokyo) 91, 1139 (1982); (184) M. Tomita, T. Osawa, Y. Sakurai, and T. Ukita, Int. J. Cancer6, 283 (1970); (185) S. D. Rosen, J. Kaur, D. L. Clark, B. T. Pardos, and W. A. Frazier, J. Biol. Chem. 254, 9408 (1979); (186) T. K. Gartner and M. L. Ogilvie, Biochem. J. 224, 301 (1984); (187) N. N. Desai and A. K. Allen, Anal. Biochem. 93, 88 (1979); (188) D. C. Kilpatrick, Biochem. J. 191, 273 (1980); (189) I. Matsumoto, A. Jimbo, Y, Mizuno, N. Seno, and R. W. Jeanloz, J. Biol. Chem. 258, 2886 (1983); (190) R. D. Por6tz, H. Riss, J. W. Timberlake, and S. M. Chien, Biochemistry 13, 250 (1974); (191) S. Shimizu, M. Ito, and M. Niwa, Biochim. Biophys. Acta 500, 71 (1977); (192) E. Cohen, G. R. Vasta, W. Korytnuk, C. R. Petrie, and N. Sharma, Prog. Clin. Biol. Res. 157, 55 (1984); (193) Quoted in Peumans et al."; (194) G. Uhlenbruck, D. Karduck, and R. Pearson, Comp. Biochem. Physiol. B 63B, 125 (1979); (195) B. A. Baldo, W. H. Sawyer, R. V. Stick, and G. Uhlenbruck, Biochem. J. 175, 467 (1978); (•96) R. H. Rice and M. E. Etzler, Biochemistry 14, 4093 (1975); (197) J. M. Brown, M. A. Leon, and J. J. Lightbody, J. lmmunol. 117, 1976 (1976); (198) W. C. Greene and T. A. Waldman, J. lmmunol. 124, 2979 (1980); (199) W. C. Greene, C. K. Goldman, S. T. Marshall, T. A. Fleisher, and T. A. Waldman, J. lmmunol. 127, 799 (1981); (200) I. Matsumoto and T. Osawa, Arch. Biochem. Biophys. 140, 484 (1970); (201) V. Horejsi and S. Kocourek, Biochim. Biophys. Acta 336, 329 (1974); (202) N. Yamaguchi, K. Oshimatsu, S. Toyoshima, and T. Osawa, J. Immunol. 126, 2290 (1981); (203) C. R. Jenkin and D. Rowley, Aust. J. Exp. Biol. Med. Sci. 48, 129 (1970); (204) C. M. Baumann, A. D. Strosberg, and H. Rudiger, Eur. J. Biochem. 122, 105 (1982); (205) C. Baumann, H. Rudiger, and A. D. Strosberg, FEBS Lett. 102, 216 (1979); (206) G. W. G. Bird and J. Wingham, J. Clin. Pathol. 34, 69 (1981); (207) N. Fornstedt and J. Porfith, FEBS Lett. 57, 187 (1975); (208) J. J. Hemperly, T. P. Hopp, J. W. Becker, and B. Cunningham, J. Biol. Chem. 254, 6803 (1979); (209) T. P. Hopp, J. J. Hemperly, and B. A. Cunningham, J. Biol. Chem. 257, 4473 (1982); (210) J. J. Hemperly, K. E. Mostov, and B. A. Cunningham, J. Biol. Chem. 257, 7903 (1982); (211) A. K. Allen, N. N. Desai, and A. Neuberger, this series, Vol. 50 [36]; (212) E. Lisowska, W. Szeliga, and M. Duk, FEBS Lett. 72, 327 (1976); (213) M. Duk and E. Lisowska, Eur. J. Biochem. 118, 131 (1981); (214) A. Falasca, C. Franceschi, C. A. Rossi, and F. Stirpe, Biochim. Biophys. Acta 577, 71 (1979); (215) G. Gebauer, E. Schiltz, A. Schimpl, and

[1]

T CELL MITOGENSAND PBA

15

References to TABLE I (continued) H. Rudinger, Hoppe-Seyler's Z. Physiol. Chem. 360, 1727 (1979); (216) L. Grubhoffer, M. Ticha, and J. Kocourek, Biochem. J. 195, 623 (1981); (217) S. E. Tollefsen and R. Kornfeld, J. Biol. Chem. 258, 5172 (1983); (218) V. L. Braciale, H. P. Friedman, and T. J. Braciale, J. Immunol. Methods 43, 241 (1981); (219) R. H. McDonald, J. D. Mach, M. Schreyer, P. Zaech, and J. C. Cerottini, J. Immunol. 126, 883 (1981); (220) S. E. Tollefsen and R. Kornfeld, J. Biol. Chem. 258, 5165 (1983); (221) C. N. Hankins and L. M. Shannon, J. Biol. Chem. 253, 7791 (1978); (222) P. Luther, H. Theise, B. Chatterjee, D. Karduck, and G. Uhlenbruck, Int. J. Biochem. 11, 429 (1980); (223) J. Y. Lin and T. B. Chou, J. Biochem. (Tokyo) 96, 35 (1984); (224) S. Sugij and E. A. Kabat, Biochemistry 19, 1192 (1980); (225) G. Cheung, A. Haratz, M. Katar, R. Skrokov, and R. D. Por6tz, Biochemistry 18, 1646 (1979); (226) P. M. Kaladas and R. D. Por6tz, Biochemistry 18, 4806 (1979); (227) P. M. Kaladas, R. Goldberg, and R. D. Por6tz, Mol. Immunol. 20, 727 (1983); (228) M. M. Roberson and S. H. Barondes, J. Biol. Chem. 257, 7520 (1982); (229) M. M. Roberson, A. P. Wolffe, J. R. Tata, and S. H. Barondes, J. Biol. Chem. 260, 11027 (1985).

incorporation o f radioactive thymidine in t r a n s f o r m e d l y m p h o c y t e s measures the extent of D N A synthesis. L e s s c o m m o n l y used p a r a m e t e r s are the m e a s u r e m e n t of the rate of protein synthesis or the rate of R N A synthesis using radiolabeled amino acids or uridine, respectively. The effect of the various c o m p o n e n t s o f the m e d i u m and conditions of incubation on the r e s p o n s e of l y m p h o c y t e s to mitogens has been detailed elsewhere in this volume [3]. See also [10] and [11]. R a t e o f P r o t e i n S y n t h e s i s . The c o m m o n l y used labeled amino acids are L-[3H]leucine or L-[3H]alanine (specific activity about 50 Ci/mmol) or L-[14C]leucine or L-[14C]alanine (specific activity about 300 mCi/mmol). M o s t culture m e d i a contain L-leucine. This amino acid has to be deleted, therefore, f r o m the m e d i u m if [3H]- or L-[~4C]leucine are used. Alanine is not present in R P M I 1640 medium. The radioactive amino acid (at a final concentration of 1 ~Ci/10/zl) is added at the beginning of the incubation. The cultures are harvested after 24-36 hr. The rest of the p r o c e d u r e is as detailed below. R a t e o f R N A S y n t h e s i s . The rate of R N A synthesis is usually m e a s u r e d b y the addition of [~4C]uridine (sp. act. 50 mCi/mmol; 0.25 /zCi/ml) or 125I-labeled deoxyuridine (sp. act. 2200 Ci/mmol) for the last 6 hr of incubation (see also this series, Vol. 116 [39]). The rest of the p r o c e d u r e is as detailed below. C o m m e n t . Bacterial contamination is the m o s t frequent p r o b l e m in this type of experiment. This can be easily avoided by filter sterilizing the reagents before use and by observing the c o m m o n rules of sterile work. Bacterial contamination is suggested by higher than e x p e c t e d and uniform radioactivity in the wells. It is confirmed by examining the cultures under an inverted microscope.

16

STIMULATION OF LYMPHOCYTES

[1]

TABLE II COMMONLY USED POLYCLONALB CELL ACTIVATORS PBA

References"

Pokeweed mitogen Dextran sulfate Polyvinylpyrrolidone Pneumococcal polysaccharide III Poly(A-U) Purified protein derivative Poly(I-C) Lipopolysaccharide Staphylococcal organisms Bacto streptolysin O reagent Staphylococcal phage lyate Epstein-Barr virus Nocardia water-soluble mitogen

1 2-6 2-5 2-6 2-5 2-6 2 2 8, 9 10 7 11, 12 13

Key to references: (1) G. Janossy, E. Gomez De La Concha, M. Waxdal, and T. Platts-Mills, Clin. Exp. lmmunol. 26, 108 (1976); (2) J. Anderson, O. Sj6berg, and G. Moiler, Eur. J. Immunol. 2, 349 (1972); (3) A. Coutinho and G. Moiler, Eur. J. lmmunol. 3, 608 (1973); (4) R. Dorris, A. Schimpl, and E. Wecker, Fur. J. Immunol. 4, 230 (1974); (5) G. Janossy and M. Greaves, Transplant. Rev. 24, 177 (1975); (6) A. S. Fauci and K. R. Pratt, J. Exp. Med. 144, 74 (1976); (7) J. H. Dean, J. S. Silva, J. L. McCoy, J. J. Baker, C. Leonard, and R. B. Herberman, J. Immunol. 115, 1060 (1975); (8) P. E. Lipsky, J. Immunol. 125, 155 (1980); (9) G. Montazeri, N. Chiorazzi, S. M. Fu, and H. G. Kunkel, J. Clin. Immunol. Immunopathol. 16, l (1980); (10) T. A. Waldmann and S. Broder, Adv. lmmunol. 32, l (1982); (11) H. Kirchner, G. Tosato, R. M. Blaese, S. Broder, and I. T. Magrath, J. Immunol. 122, 1310 (1979); (12) G. Tosato, I. T. Magrath, I. R. Koski, N. J. Dooley, and R. M. Blaese, J. Clin. Invest. 66, 383 (1980); (13) C. Bona, S. Broder, A. Dimitriv, and T. A. Waldmann, lmmunol. Rev. 45, 69 (1979).

T r e a t m e n t o f D a t a . T h e e x t e n t o f the m i t o g e n i c r e s p o n s e o f l y m p h o c y t e s is u s u a l l y e x p r e s s e d as the s t i m u l a t i o n i n d e x (SI),

SI = (cpm~xp - cpmblank)/cpmbl~nk w h e r e cpm~xp a n d cpmbl~k are the c p m i n c o r p o r a t e d b y the cells i n c u b a t e d in the p r e s e n c e a n d in the a b s e n c e o f m i t o g e n , r e s p e c t i v e l y . I n T a b l e I, the b e s t k n o w n l e c t i n s o f v e g e t a b l e a n d a n i m a l origin are

[2]

D E R I V A T I V E S OF C O N C A N A V A L I N A

17

listed together with key references to their purification and biochemical characterization, carbohydrate specificity, and mitogenicity. Polyclonal B Cell Activators Polyclonal B cell activators (PBA) are substances that stimulate B cell proliferation and promote their differentiation into antibody-producing plasma cells. An excellent review on this subject has appeared recently. 10 Table II, modified from Ref. 10, lists the more commonly used PBAs. The activity of PBAs may be assessed by determining the rate and extent of B cell proliferation in systems analogous to those detailed above for T cells (see below) or by measuring the production of Ig. These methods have been preserved elsewhere in this series (Vol. 73 [38-46]; Vol. 116 [1-7]) and in this volume [3] and will not be duplicated here. Both T cells and macrophages (monocytes) play an important role in regulating the response of B cells to PBAs. Methods have been presented elsewhere in this series for the depletion of T cells (Vol. 108 [7, 15-19, 21]) and macrophages (Vol. 108 [26-32]). The effect of the various components of the medium and conditions of incubation on the response of lymphocytes to mitogens has been detailed elsewhere in this volume [3]. ~0T. A. Waldman and S. Broder, Adv. Immunol. 32, 1 (1982).

[2] C r o s s - L i n k e d D e r i v a t i v e s o f C o n c a n a v a l i n A

By TOSHIAKI OSAWA and MASATOSHI BEPPU Introduction Lectins have multiple saccharide-binding sites; therefore it has been assumed that receptor cross-linkages may play an important role in some of their effects on cells, such as the triggering of lymphocyte mitogenesis and receptor rearrangements. 1-3 Concanavalin A (Con A) is the most widely used lectin in immunology and cell biology. It activates T M. Greaves and G. Janossy, Transplant Rev. 11, 87 (1972). 2 G. L. Nicolson, Int. Rev. Cytol. 39, 89 (1974). 3 H. Lis and N. Sharon, in "The Antigens" (M. Sera, ed.), Vol. 4, p. 429. Academic Press, New York, 1977.

METHODS IN ENZYMOLOGY, VOL. 150

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.