Mitogen induced cytotoxicity in the nurse shark

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 5, pp. 53-64, 1981 0145-305X/81/010053-12502.00/0 Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

MITOGEN INDUCED CYTOTOXICITY IN THE NURSE SHARK1

Carolyn L. Pettey and E. Churchill MeKinney Department of MicrobiologyD4-4 University of Miami School of Medicine P.O. Box 016960 Miami, Florida 33101

ABSTRACT

Nurse shark (Ginglymostoma cirratum) leukocytes isolated from peripheral blood were demonstrated to possess cytotoxic activity towards xenogeneic marine erythrocyte targets (grey snapper) when stimulated by phytohemagglutinin,eoneanavalin A, or lipopolysaeeharide. These leukoeytes when separated on the basis of adherence to glass exhibited a differential cytotoxic effect. Only the adherent population showed significant cytotoxicity toward the target cells in the presence of any of the mitogens. Wright-Giemsastaining showed some differences in the two populations, however, both adherent and nonadherent cell populations contain all types of leukocytes. Upon surface marker analysis, the adherent cells were found to be depleted of sheep erythrocyte rosette forming cells and surface immunoglobulin positive cells compared to the nonadherent and unseparated populations. The cytotoxic reaction in the shark resembles that of mammalian species and the effector leukocyte(s) may be the phylogenetie precursor of mammalian cytotoxic effector cells. INTRODUCTION

The cellular immune system of lower vertebrates has been analyzed to elucidate when lymphoeytes diverged in phylogeny. In teleost fish, evidence has been presented for the existence of ceils analogous to mammalian T and B lymphoeytes(1-4). Sharks, which are cartilagenous fish, have been shown to differ from teleosts in several

1Supported in part by the Lerner Fund for Marine Research of the American Museum of Natural History and by Biomedical Research Support Grant RR0536318 from the Biomedical Research Support Branch, NIH.

53

54

CYTOTOXICITY

IN SHARKS

Vol.

5, No. 1

immunologic processes. Allograft rejection occurs rapidly in higher teleosts (5,6) while this same response takes place in a chronic manner in sharks (7). Sharks, in contrast to teleost fish, are reported to have a poor or undetectable anamnestic antibody response (8). These data suggest that the immune system in the shark may be less advanced than that of the teleost fish. The shark, therefore, may provide a suitable model for the investigation of lymphocyte diversity in a more primitive system. Previous reports concerning in vitro blastogenic studies in the nurse shark (Gin~lymostoma cirratum) have shown minimal responses to various mitogens (9). We have earlier described a mitogen induced cytotoxic system using the same shark species where we obtained responses comparable in magnitude to mammalian systems (I0). Our current research was aimed at further defining the cells involved in mitogen induced cytotoxicity in the shark. This was accomplished by using T and B lymphocyte mitogens, performing cell separations and analyzing the leukocytes by morphology and cell membrane markers. MATERIALS AND METHODS Collection of blood. Nurse sharks were anaesthetized in a solution of 1 part per million of tricaine methane sulfonate in sea water. Anaesthetized sharks were bled from the caudal v e i n . Blood was mixed with sodium heparin to give a final concentration of 100 units/ml. Medium. The basic culture medium for all shark leukocytes was RPMI-1640 (GIBCO) adjusted to contain 0.2 M NaCl and 0.35 M urea (shark medium) which is isotonic for shark cells. Shark medium contained antibiotics (100 units/ml penicillin, 100 ug/ml streptomycin, and 0.25 ug/ml fungizone) and 10% heat-inactivated fetal bovine serum. HEPES buffer (25 mM) was used to maintain pH. All other salt solutions and media were adjusted to shark isotonicity with additional NaCl and urea. Leukocyte isolation. To remove erythrocytes from the shark blood, whole blood was layered onto a mixture of 2 parts of Lymphocyte Separation Medium (Bionetics Inc.) mixed with 1 part 0.85% saline. This dilution permitted the red blood cells (RBC) to pellet and the entire leukocyte popula~on remained at the interface upon centrifugation at 400 x g for 40 minutes at 18-20 . Leukocytes were removed from the interface and washed in shark medium prior to further use. Mitogens. Stock solutions of mitogens were prepared in the following concentrations: phytohemagglutinin-P (PHA, Difco) 2.5 mg/ml, concanavalin A (Con A, Calbiochem) 1 mg/ml, and lipopolysaccharide (LPS, E. coli 055:B5, Difco) I0 mg/ml. Further dilutions were made in shark medium. Mitol~en induced cytotoxicity. The cytotoxicity assay used was based on that of Perlmann and Perlmann (11). In addition to shark RBC, grey snapper (Lutjanus griseus) RBC were employed as target cells due to their ab~l'l~V to tolerate shark medium. Briefly, 1.25 x 10- leukocytes were added to 5 x 10---Cr-labelled target cells in a total volume of 540 ul of shark medium with or without mitogen. Mitogens were added in 40 ul quantities to provi.~ the appropriate final concentration. To ~ccount for spontaneous release of the v-Cr label, control cultures contained 5 x I0 v unlabelled snapper or shark RBC in pla~e of leukocytes. Cultures were incubated in tightly capped tubes for 18 hr at 30 and terminated by centrifugation at 400 x g for 10 minutes. Results were obtained by separately counting the pellet and the supernatant in a gamma counter. Calculations were as follows: %51Cr release = counts per minute (cpm) of the supernatant x i00 cpm of the supernatant plus the pellet % eytotoxicity = %51Cr release of the experimental - % 51Cr release of the control

Vol. 5, No. i

CYTOTOXICITY

IN SHARKS

55

Cell separation by adherence to glass. The technique employed made use of a glass bead column as described by Shortman et al. (12) except all solutions were adjusted to contain 0.2 M NaCI and 0.35 M urea. Siliconized glass beads of a300-600 u diameter were used. After equilibrating and washing the column, 200 x 10Vcells were applied. The nonadherent cells were washed through; the adherent cells were eluted with an EDTA buffer and shaking the column. All cells were washed prior to further use. E-rosette formation. Erythrocyte (E) rosettes were performed utilizing neuraminidase-treated sheep RBC according to the method of Galili and Sehlesinger (13). At least 200 leukocytes were counted and results expressed as % rosetting cells. Leukocytes with three or more adherent RBC were counted as rosettes. Surface immunoglobulin (Slg) determination. Purified 19s shark IgM, donated by Dr. L. Fuller (University of Miami, ~iami, FI), was used to immunize rabbits. Rabbits were immunized with the 19s IgM in complete Freund's adjuvant subcutaneously at multiple sites. After the rabbits exhibited a titer, they were boosted. Titers were determined using Ouchterlony double diffusion in agar (14). Booster injections consisted of 19s IgM in incomplete Freund's adjuvant administered subcutaneously. Serum obtained from immunized rabbits was heat inactivated at 56 for 1 hr. Adsorption of the ro~bbit serum was done twice with washed shark RBC. Shark leukocytes (2-3 x 10 ) which have been washed once wlth 0.03 M NaN~ in Hanks balanced salt solution adjusted to shark isotonicity were used for staining7 Approximately 50 ul of antiserum was added and incubated for 1 hr at 23v (room temperature). The leukocytes were washed twice with the azide solution and 50 ul of appropriately diluted fluorescein isothiocyanate conjugated goat anti-rabbit immunoglobulin was added and incubated for 1 hr at 4% Normal rabbit serum and the fluoreseein conjugated antibody alone served as controls. The leukoeytes were washed twice more with the azide solution and examined using fluorescent microscopy (Leitz phasefluorescent microscope equipped with epifluorescent attachments). Results are expressed as % cells with membrane fluorescence. .u~

.

.

•

,

Wright-Giemsa staining. Leukocytes were smeared onto glass slides and almost allowed to dry. One ml of Wright's stain (Harleco, Lot #7300) was added to the slides for 3 minutes. The slides were then covered with 1.0 ml of phosphate buffer, pH 6.7, which was allowed to remain for 10 minutes and poured off. One ml of Giemsa stain (Fisher, Lot #742675, diluted I:I0 with distilled water) was added, reacted for 10 minutes and the slides decolorized with distilled water. Slides were allowed to air dry prior to microscopic examination. Statistics. variates.

Statistical analyses were performed using the Student's t test for paired

RESULTS Dose dependent eytotoxic response with mitogens. Shark ]eukocytes were stimulated with various concentrations of each of the three mitogens, PHA, Con A, and LPS, and assayed for cytotoxic reactivity against xenogeneic marine erythroeytes. Each shark tested responded to the mitogens in a dose dependent fashion. Typical responses are depicted in Figure 1. The optimal PHA and Con A concentrations were fairly consistent from shark to shark with an average PHA optimum of I00 ug/eulture and an average Con A optimum of 40 ug/culture. The LPS optimum, however, varied widely between animals with optima ranging from 25 to 400 ug/culture. The LPS concentration chosen for use was 100 ug/culture.

56

CYTOTOXICITY IN SHARKS

Vol. 5, No. I

70

60 1

50

t

~

~4o

LPS

•

Con A 0 PHA

1I/ / lo

0

I

2,5

t

50

I

75

t

100

I

125

I

150 ~ g OF MITOGEN/CULTURE

I

t

I

175

200

225

250

Figure 1 Dose dependent eytotoxic response to PHA, Con A and LPS. E a c h curve represents a typical response of a shark. These data show a dose dependent response to mitogen used.

The cytotoxie response of shark leukocytes in the presence of optimal concentrations of each of the three mitogens was compared with the response of shark leukocytes in the presence of the same amount of shark medium. The effector to target cell ratio was 25:1, which was previously determined to be optimal as compared with ratios of 50:1 and 100:1. As shown in Table 1, shark leukoeytes had a significant response against the target, snapper RBC, only when stimulated by any of the three mitogens.

Vol. 5, No. I

CYTOTOXICITY IN SHARKS

57

50

FFFF]GROWTH MEDIUM 45

F~PHA FFJTJ CON A

40

F-] LPS

35 >-

30 (.; X O

25 O >U

20

15 T-

10

5

i

llnml

IIIII unseparated

....

]

I lllll

I

nonadherent CELL POPULATIONS

IIII] ISlII III11 IlllJ Illll adherent

Figure 2 Cytotoxic responses of glass adherent and nonadherent leukoeytes. The results depicted are the averages of 9 separate experiments; each experiment performed in triplicate. The adherent cell population, which represented 30% of the total leukocytes recovered, contained the majority of the cells responding to the mitogens. The nonadherent cell population possessed virtually no cytotoxic reactivity.

58

CYTOTOXICITY IN SHARKS

Vol. 5, No. 1

TABLE 1 Cytotoxic Effects of Shark Leukocytes From 5 Representative Experiments

Experiment Number

No Mitogen

PHA

1

1.6 a

14.3

13.7

8.8

2

6.3

24.1

17.2

17.1

3

0.5

22.4

7.2

22.8

4

0

22.5

2.8

12.2

5

4.5

32.8

19.7

23.0

Con A

LPS

Percent cytotoxicity. All of the mitogens tested produced a significant cytotoxic response (p < .02).

TABLE 2 Cytotoxicity of Shark Leukocytes toward Snapper or Shark RBC Targets

Target RBC Snapper Experiment Number

Shark a

No Mitogen

PHA

No Mitogen

1

0.1 b

6.3

0

0

2

1.5

29.3

0

0

3

0.5

19.1

0

0

4

3.3

19.0

0

0

5

1.4

37.0

0

5.2

PHA

Autologous or allogeneic RBC. Percent cytotoxicity. A significant cytotoxic response was seen only against snapper, or xenogeneic RBC in thepresence of PHA (p <.02).

Vol. 5, No. I

CYTOTOXICITY IN SHARKS

59

Target cell restriction. To determine whether the cytotoxic response of shark leukocytes was limited to xenogeneic target cells, assays utilizing allogeneie or autologous target cells were performed. PHA was chosen as the stimulating mitogen. As can be seen in Table 2, leukocytes stimulated with PHA were not able to lyse either autologous or allogeneic target RBC, while having an effect on the xenogeneic target RBC. Cytotoxic responses of glass adherent and nonadherent cells. The nonadherent cell fraction comprised approximately 70% of the recoverable cells; the adherent, 30%. These two cell populations were assayed for cytotoxic reactivity. The glass adherent population was found to contain virtually all of the cells responsive to the three mitogens, as illustrated in Figure 2. Lack of cytotoxieity in the nonadherent cells was not due to a loss of viability as judged by trypan blue exclusion. Morphology of glass adherent and nonadherent populations. The leukocytes of both nonadherent and adherent populations were examined following Wright-Giemsa staining. For convenience in cell counting and quantitation, the cell types were grouped morphologically as follows: lymphoeytes, monocytes, thrombocytes, blast cells, and granuloeytes. As depicted in Table 3, both cell populations contained leukocytes of all types. The nonadherent population, however, was enriched with lymphocytes, whereas the adherent population was increased with respect to monoeytes, blast cells, and granulocytes. Morphological identification did not provide a method of clearly differentiating adherent and nonadherent populations.

TABLE3 Comparison of Nonac~erent and Adherent Cell Populations by Wright-Giemsa Staining Adherent

Nonadherent

lymphocyte

12.0 + 3.3 a

41.0 +_ 1.2

monocyte

38.3 +_ 4.5

27.0 _+ 0.8

thrombocyte

20.7 _+ 1.2

14.7 + 1.5

blast cell

17.7 + 0.9

7.7 + 0.7

D

granuloeyte

12.3 + 3.5

6.3 + 0.9

Expressed in percentage + standard error. The results expressed are from 4 separate experiments.

60

CYTOTOXICITY

IN SHARKS

Vol. 5, No.

Membrane markers. Rabbit antiserum prepared against purified shark 19s IgM was used to stain shark leukocytes by indirect immunofluorescence. Unadsorbed antiserum was found to stain all leukocytes and contaminating RBC. Following adsorption with shark RBC, the nonspecific staining of erythrocytes was removed, but not that of certain leukocytes. The presence of Slg was determined for unseparated, nonadherent, and adherent leukocytes using the adsorbed rabbit anti-shark 19s IgM. A differential staining effect was observed, as seen in Table 4. The nonadherent population contained 32% fluorescing cells while the adherent population contained only 9%. The majority of SIg-bearing cells were small, round cells which had the appearance of lymphocytes when viewed under phase-contrast microscopy.

TABLE 4 Surface Immunoglobulinon Shark Leukocyte Populations Cell Population

a

Unseparated

Nonadherent

Adherent

19.2 + 1.4 a

31.6 + 4.2 b

9.1 + 1.7

Expressed in percentage + standard error. average of 6 separate exper-iments.

T h e s e results represent the

The percentage of Slg bearing cells is significantly increased (p < .02) in the nonadherent population as compared with the adherent and unseparated populations.

E rosette formation of shark leukocytes was investigated using human, rabbit, snapper, and sheep RBC. Shark leukocytes formed E rosettes only with neuraminidase treated sheep RBC. As depicted in Table 5, the nonadherent cell population contained the majority of the sheep E rosetting cells. Using phase-contrast microscopy, there appeared to be two cell types which form E rosettes--one resembling a small lymphocyte, the other a granulocyte.

1

Vol. 5, No. i

CYTOTOXICITY

IN SHARKS

61

TABLE 5

E Rosetting Capacity of Shark Leukocyte Populations Cell Population

a

Unseparated

Nonadherent

Adherent

6.6 + 1.6 a

9.0 + 2.2 b

3.1 + 0.6

Expressed in percentage + standard error. These results are the averages of 6 separate experiments. The nonadherent population contained significantly more rosetting cells (p < .02) than the adherent.

DISCUSSION Shark leukocytes can be stimulated to kill xenogeneic target ceils by three mitogens. Concentrations of T cell mitogens were similar to those used for mammalian leukocytes (15). In higher vertebrates, the B cell mitogen LPS is not commonly used to elicit a cytotoxic response; however, the concentration used per cell in the shark was similar to concentrations utilized to induce blast transformation in mammals (16). Two of the mitogens, which induced cytotoxicity (PHA and Con A), were previously used by Lopez et al. (9) to induce blast transformation in shark leukocytes~ The concentrations of Con "A required to elicit a response were very large (I mg/10 v cells) as c~mpared with what is necessary to induce a response in mammalian systems (i0 ug/10 v cells). They postulated this to be due to differences in the receptor densities between mammalian and shark cells. In contrast, our data show that the concentrations of Con A required to induce cytotoxicity in cells from both sharks and mammals do not differ greatly, suggesting that these vertebrates have similar receptor densities. T h i s could be due, however, to differences between the mechanism of activation for cytotoxicity and blastogenesis (17,18). In addition, Lopez et al. found that PHA did not stimulate any blastogenic response in unseparated leukocyte populations, but did stimulate a response in a separated cell population. It was postulated that this was due to the presence of suppressor cells. No such suppressive effect on PHA induced cytotoxicity was found in our experiments. We have demonstrated target cell specificity in the shark cytotoxic reaction. Autologous or allogeneic target cells were not affected by the stimulated shark leukocytes while xenogeneic cells were lysed. This is in contrast to mitogen induced cytotoxic reactions in higher vertebrates where PHA stimulated leukocytes lyse autologous targets as readily as foreign targets (19,20). Target cell recognition occurs in higher vertebrates in natural killer systems (21); a similar type of recognition process may be occurring in the shark leukocytes when stimulated by mitogens. Thus, the mitogen stimulated response appears to be less target cell specific in higher vertebrates than in the shark. These data also show that mitogen stimulated shark leukocytes are able to distinguish autologous or allogeneic targets from xenogeneie targets.

62

CYTOTOXICITY IN SHARKS

Vol. 5, No. 1

Although this reaction demonstrates a non-self recognition of target cells that is not associated with this response in mammalian systems, the mitogen induced cytotoxic response is similar to that of higher vertebrates in regards to mitogen concentrations, effector to target cell ratio, magnitude of response, and length of incubation. In mammals, a variety of leukocytes have been implicated in mitogen induced cytotoxic responses against erythrocyte targets including monocytes, granulocytes and lymphocytes (22). The shark effector cell population contains leukocytes which morphologically resemble all the mammalian effector cells; however, these leukoeytes are also present in the non-effector cell population. Glass adherence, used to isolate shark effector leukocytes, is a characteristic of most monocytes and granulocytes of higher vertebrates. Thus the activity of adherent shark cells may more closely resemble that of mammalian monocytes and granulocytes rather than lymphocytes. Surface marker data further suggests that the primary effeetor cell may not be the lymphocyte. Most leukocytes that formed E rosettes or that showed SIg were in the non-effector population. The nonadsorbed rabbit serum first used to stain for the presence of Slg possessed reactivity with membranes of shark erythrocytes. This reactivity may be due to rabbit antibody made against the carbohydrate moities of shark immunoglobulin. Researchers studying the leukocytes of other lower vertebrates have found a similar cross-reactivity in antibodies produced in rabbits against the purified high molecular weight immunoglobulins of the trout and frog (23,24). While the differences in the cytotoxic responses between sharks and mammals may represent divergent evolution, the glass adherent cytotoxic effector cell in mammals may have evolved from a system similar to the sharks'. One cell type in the shark may be responsible for cytotoxic reactions mediated by more than one cell population or subpopulation in mammals. T h i s shark leukocyte population may represent the phylogenetic"precursor of the effector cells responsible for mitogen induced cytotoxicity and natural killer activity in mammalian systems.

ACKNOWLEDGMENTS We thank Mantley Dorsey, Jr. for his expert technical assistance in handling the animals and the Miami Seaquarium for providing the animals and their maintenance. REFERENCES 1.

ETLINGER, H. N., HODGINS, H. O. and CHILLER, J. J. Evolution of the lymphoid system I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. J. Immunol. 116, 1547, 1976.

2.

YOCUM, D., CUCHENS, M. and CLEM, L. W. The hapten-carrier effect in teleost fish. J. Immunol. 114, 925, 1975.

.

RUBEN, L.N., WARR, G. W., DECKER, J. M. and MARCHALONIS, J. J. Phylogenetic origins of immune recognition: Lymphoid heterogeneity and the hapten/carrier effect in the goldfish, Carassius auratus. Cell. Immunol. 31, 266, 1977.

4.

CUCHENS, M. A. and CLEM, L. W. Phylogenyof lymphoid heterogeneity If. Differential effects of temperature on fish T-like and B-like cells. Cell. Immunol. 34, 219, 1977.

Vol. 5, No.

1

CYTOTOXICITY

IN SHARKS

63

5.

HILDEMANN, W. H. Scale homotransplantation in gold fish (Carassius auratus). Ann. N.Y. Acad. Sci. 64, 775, 1957.

6.

MARCHALONIS,J. J. Immunityin Evolution. Harvard, Cambridge: University Press, 1977.

7.

BORYSENKO, M. and HILDEMANN, W. H. Reactions to skin aUografts in the horn shark. TransDl. 10, 287, 1970.

8.

CLEM, L. W. and SMALL, P. A. Phylogenyof immunoglobulin structure and function. J. Exp. Med. 125, 893, 1967.

9.

LOPEZ, D. M., SIGEL, M. M. and LEE, J. C. Phylogenetie studies on T cells I. Lymphocytesof the shark with differential response to phytohemagglutinin and concanavalin A. Cell. Immunol. 10, 287, 1974.

I0.

McKINNEY,E. C., PETTEY, C. L., LOPEZ, D. M. and SIGEL, M. M. Cytotoxicity of nurse shark (Ginglymostomaeirratum) leukocytes. In: Developmental Immunobiology. J. B. Solomon and J. D. Horton (Eds). Amsterdam; Biomedical Press, 1977, p. 217.

11.

PERLMANN,P. and PERLMANN, H. 51Cr-release from chicken erythrocytes: An assay system for measuring lymphocytes in vitro. In: In Vitro Methods in Cell-MediatedImmunity. B. R. Bloom and P. R. Glad-e New York:Academic Press, 1971, p. 361.

12.

SHORTMAN, K., WILLIAMS, H., JACKSON, H., RUSSELL, P., BYRT, P. and DIENER, E. The separation of different cell classes from lymphoid organs IV. The separation of lymphocytes from phagocytes on glass bead columns and its effect on subpopulations of lymphoeytes and antibody-formingcells. J. Cell Biol. 48, 506, 1971.

13.

GALILI, U.and SCHLESINGER, M. The formation of stable E rosettes after neuraminidase treatment of either human peripheral blood lymphocytes or of sheep red blood cells. J. Immunol. 112, 1628, 1974.

14.

OUCHTERLONY, O. In: Progress in Allergy. P. Kallosand B. H. Waksman(Eds). BasehKarger, 1962, p. 30.

15.

WISLOFF, F. and FROLAND, S. S. Characterization of subpopulations of human lymphoid cells participating in phytohemagglutininand concanavalin A induced cytotoxicity. Int. Arch. Allergy 45, 456, 1973.

16.

MILLER, R. A., GARTNER, S. and KAPLAN, H. S. Stimulationof mitogenie responses in human peripheral blood lymphocytesby lipopolysaccharide: Serum and T helper cell requirement. J. Immunol. 121, 2160, 1978.

17.

PERLMANN, P., NILSSON, H. and LEON, M.S. Inhibition of cytotoxicity of lymphoeytesby coneanavalin A in vitro. Science 168, 112, 1970.

18.

DAYNES, R. A. and GRANGER, G. A. The regulation of lymphotoxin release from stimulated human lymphocyte cultures: The requirement for continual mitogen stimulation. Ceil. Immunol. 12, 252, 1974.

19.

LUNDGREN, G. and MOLLER, G. Non-specific induction of eytotoxieity in normal human lymphocytes in vitro: Studies of mechanism and specificity of the reaction. Clin. Exp. Immunol. 4, 435, 1969.

64

CYTOTOXICITY

IN SHARKS

Vol. 5, No. I

20.

PERLMANN, P., PERLMANN, H. and HOLM, G. Cytotoxic action of stimulated lymphocytes on allogenic and autologous erythrocytes. Science 160, 306, 1968.

21.

HERBERMAN, R. B., NUNN, M. E. and LAVRIN, D. H. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneie and allogeneic tumors. I. Distribution of reactivity and specificity. Int. J. Cancer 16, 216, 1975.

22.

NELSON, D. L., BUNDY, B. M., PITCHON, H. E., BLAESE, R. M. and STROBER, W. The effector cells in human peripheral blood mediating mitogeninduced cellular cytotoxicity and antibody-dependentcellular cytotoxicity. J. Immunol. 117, 1472, 1976.

23.

YAMAGA, K. M., KUBO, R. T. and ETLINGER, H. M. Studieson the question of conventional immunoglobulinof thymocytesfrom primitive vertebrates. II. Delineation between /g-specific and cross-reactive membrane components. J. Immunol. 120, 2074, 1978.

24.

MATTES, M. J. and STEINER, L. A. Surface immunoglobulinon frog lymphocytes. Identification of two lymphocytepopulations. J. Immunol. 121, 1116, 1978.