The characterization of the toad splenocytes which bind mouse anti-human IL-2 receptor antibody

Immunology Letters, 16 (1987) 43-48

Elsevier IML 00928

The characterization of the toad splenocytes which bind mouse anti-human IL-2 receptor antibody Lorene Langeberg 1, Laurens N. R u b e n 1, Richard H. Clothier 2 a n d Stanley Shiigi 3 IDepartment of Biology, Reed College, Portland, Oregon, U.S.A.; 2Department of Human Morphology, Queen'sMedical Centre, University of Nottingham, U.K.; 3Division of Metabolic Diseases, Oregon Regional Primate Research Center, Beaverton, Oregon, U.S.A.

(Received 29 June 1987; accepted 7 July 1987)

1. Summary

We have utilized two depletion protocols to characterize the spleen cells of the South African clawed toad, which bind specifically mouse-antihuman IL-2 receptor (anti-Tac) antibody and recombinant DNA produced IL-2. When the selectively lymphotoxic reagent, N-CH3-N-nitrosourea (NMU), is injected into adult toads, it removes the thymic cortex and lymphocytes throughout the body required for helper and cytotoxic cell functions. We have also used a monoclonal mouse anti-Xenopus IgM antibody to deplete toad splenocyte populations of surface (s) Ig + cells. Freshly biopsied and cultured spleen cells were compared with respect to their capacity to bind fluorescent (FI~) anti-Tac antibody and its ligand, rIL-2, after a depletion protocol. The results clearly show that a phytohemagglutinin (PHA)/IL-2 sensitive splenocyte population is removed by NMU injection. While many of the remaining NMU insensitive, previously cultured cells are Tac +, they fail to bind rIL-2. Freshly biopsied spleen cells with constitutive IL-2 receptors are found in both the sIg- (T cell enriched) and sIg + (B cell enriched) populations fol-

Key words: IL-2 receptor; rlL-2; Binding;Amphibian; Spleno-

cyte Correspondenceto: LaurensN. Ruben, Departmentof Biology,

Reed College, 3203 SE WoodstockBlvd., Portland, OR 97202, U.S.A.

lowing panning. Moreover, both populations are able to bind the ligand with equal efficiency. Thus, constitutive IL-2 receptor bearing ceils are not restricted to either the T or B cell populations. The predominant P H A activatable, rlL-2 binding cell populations of the toad appear to be T cells which are involved with helper and cytotoxic functions.

2. Introduction

In vivo injection of human IL-2 has been used successfully to modulate immune behavior in the South African clawed toad, Xenopus laevis. This reagent will amplify reactivity to sub-immunogenic challenges with TNP-Ficoll [1]. Human IL-2 can also be used to substitute for the requirement for thymic presence in responses to that immunogen in this species [2]. Moreover, recombinant DNA produced human IL-2 (rIL-2) will substitute for carrier priming of helper function in a thymus-dependent antihapten response [3] and can provide a signal for breaking hapten-specific tolerance in adult Xenopus [4]. This ligand will specifically bind Xenopus splenocytes at sites which are visualizable with fluorescent (FI*) mouse-anti-human IL-2 receptor (anti-Tac) antibody [5]. Freshly biopsied toad splenocytes are Tac + i.e., they possess constitutive anti-IL-2 receptor antibody binding sites (IL-2R), some of which will bind rlL-2. Mitogenic stimulation with phytohemagglutinin (PHA) will not only increase the number of IL-2R/cell, but will also en-

0165-2478 / 87 / $ 3.50 © 1987 ElsevierScience Publishers B.V.(BiomedicalDivision)

43

large the number of cells capable of binding the antireceptor antibody and rIL-2. While constitutive IL-2R have been reported for mammalian immature thymic lymphocytes [6] and for natural killer (NK) cells [7], in general, IL-2 receptor expression requires immunogen or lectin activation [8]. It should be noted that while toad splenocytes may be Tac +, their immune behavior is unaffected by in vivo injection of rIL-2 in the absence of immunogen or lectin (e.g., [3]). Thus, there appears to have been a considerable degree of evolutionary conservation with respect to this ligand and its receptor. This report is part of an ongoing series of studies which have been directed at exploring the evolution of the immune regulatory network. In it, we characterize sub-populations of toad splenocytes which bind the anti-human IL-2 receptor antibody and rIL-2. To accomplish this, we have selected one protocol which will deplete the splenocyte population of specific (T) cellular functions and another which will remove sIg + (B) cells. N-CH3-N-nitrosourea, when injected in vivo, will serve as a selective lymphotoxic reagent in Xenopus which will destroy the cortex of the thymus entirely and eliminate helper and cytotoxic T cell functions within this animal [9]. The medullary region of the thymus appears to have normal cellularity. Responsivity to TNP-Ficoll and TNP-LPS, as well as thymus inducer suppressor function to thymusdependent immunogens, remain intact after treatment with this alkylating agent. The other depletion protocol involves the generation of a monoclonal mouse-anti-Xenopus IgM antibody which can be used to pan out Xenopus sIg + cells. Thus, specifically depleted freshly biopsied and/or cultured spleen cell populations of the toad are tested with respect to their capacity to bind the anti-Tac + antibody and rIL-2. Our goal is two-fold. We wish to further classify the in vitro and in vivo PHA immunogen activatable cells which have high binding capacity to rIL-2 with regard to function and to determine if the cells with constitutive IL-2 receptors are restricted to either the T or B cell populations.

44

3. Materials and Methods

3.1. Animals The toads used in all of the experiments reported here were either purchased (NASCO, Ft. Atkinson, WI) or bred in the laboratory. They were maintained at 23 °C in chlorine-free water and fed twice weekly with beef liver or prepared frog food. They were all young adults of between 10-15 g body weight and were not isogeneic animals. 3.2. Culture protocols Dissociated toad splenic lymphocytes were isolated on Histopaque (Sigma, St. Louis, MO) which was adjusted to a density of 1.09 by mixing Histopaque 1077 and Histopaque 1119. These lymphocytes were cultured (1 × 106 cells/ml) for 3 days in a medium of 60% L-15 (GIBCO, Grand Island, NY), 30% twice glass-distilled water and 10% decomplemented fetal calf serum (FCS), with or without 2/~g/ml PHA (Sigma). The binding protocols have been described in detail previously [5]. 3.3. NMU protocol The NMU was kindly supplied to RHC by Dr. Peter Swann (Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London, UK). The animals were injected intraperitoneally with a solution prepared by adding NMU to a preweighed multidose vial. The vial was then sealed, reweighed and stored in the dark at -20°C. It was dissolved in the required volume of aqueous potassium dihydrogen phosphate solution (pH 4.5) immediately before use. These preparations were done under negative pressure under strict safety precautions [9] in a glove box (A. Gallenkamp & Co. Ltd., London, UK). After injection, the animals were placed in a small volume of water for 6 h to recover. The loss of injected NMU is reduced at this stage by putting them into a small volume of water (50 to 250 ml depending on animal size). After 6 h, the water and container in which the animals had been maintained while recovering was detoxified with 3:1 10%0 sodium thiosulfate in phosphate buffer (pH 8.5). It was stored for a week, then immersed, for a further week, in a solution of potassium permanganate. It was then discarded after washing. The animals were maintained normally until they were utilized 8 days after injection of the NMU.

3.4. The mouse monoclonal anti-Xenopus IgM Ten Balb/c mice (Simonsen Labs., Gilroy, CA) were immunized intraperitoneally (i.p.) with 7.5/~g Protein-A-Sepharose CL-4B purified Xenopus laevis Ig in complete Freund's adjuvant on days 1, 43 and 57. The final series of injections on days 84 and 105 were of 100/zg purified Xenopus IgM in PBS, given either i.p. or i.v. Three days later, 2.8 × 108 splenocytes removed from three of the immunized mice were fused with 1.8 × 108 FO mouse myeloma cells [10] using PEG 4000 (BDH 9494 352E). The protocol that we used was provided by Dr. T. Tittle of the Oregon Health Sciences University, Portland, OR. After fusion, 1.5 × 107 peritoneal exudate cells were added and the cellular suspension, in a HAT/Iscove's medium, was distributed into 10 Costar 96 well plates, 0.2 ml/well. The HAT/Iscove's medium was replaced twice weekly. By day 7, approximately onethird of the wells had colonies growing in them. Culture supernatant fluids were assayed on day 11 by ELISA. Selected positive cultures were cloned twice by limiting dilution. Bulk culture of hybridomas in 175 cm 2 Falcon tissue culture flasks or ascites production in Balb/c mice yielded antibody stocks that were purified by elution from a Protein-ASepharose CL-4B column using 3 M sodium thiocyanate. Monoclonal antibody 8E4 is specific for Xenopus laevis IgM and does not recognize Xenopus borealis Ig. This suggests that this is an anti-allotype reagent. Experiments to further characterize this antibody and the 13 other monoclonals we have generated are planned. This reagent was isotyped as an IgG~, kappa antibody by ELISA. 3.5. The panning protocol Lymphocytes were depleted of slg ÷ cells using a panning technique. Bacteriological grade Petri dishes were coated with 3 ml of Protein-A-Sepharose purified monoclonal antibody 8E4 at a concentration of 200 tzg/ml in Tris-HC1 buffer at pH 8.5 for 1 h at room temperature. Non-adherent antibody solution was recovered, sterile filtered and stored at 4 oC. The coated dish was washed twice with sterile 20 mM PBS at pH 7.2 and once with sterile PBS with 1070 FCS. These dishes were either used immediately or stored upside down at - 2 0 °C for up to 1 wk. Sterile PBS with 5070 FCS was added immediately to dishes upon their removal from the freezer. All PBS was re-

moved from the dishes prior to the addition of 1.1 x 107, or fewer, lymphocytes in 1.1 ml L-15 complete medium with 5°70FCS. A dish with the cells was incubated for 60 min at 4 °C on a flat surface. At 20 min intervals, the dish was removed and rocked slowly. After 1 h, the non-adherent cells were removed and the plate was gently washed with 3 ml of cold L-15 with 5070 FCS. The cells which were freed by this wash were pooled with the non-adherent lymphocyte population. Adherent lymphocytes were recovered after vigorous pipetting with L-15 medium containing 507oFCS at 37 °C. All recovered cells were washed and aliquots of them were counted. Lymphocytes which had been separated by panning on monoclonal mouse anti-Xenopus laevis IgM coated dishes were tested with an indirect fluorescent antibody binding assay in order to establish the efficacy of the depletion protocol. Briefly, individual populations of unpanned, non-adherent and adherent lymphocytes (5 × 105) were incubated with either 50/zl PBS with 5070 FCS plus 0.1070 sodium azide, 50 #1 of a 1:10 dilution of monoclonal antibody 8E4 ascites fluid or 50/~1 of undiluted ascites fluid for 1 h at 4 °C. These cells were then washed twice prior to incubation with 50 #1 FITC-F(ab')2 sheep anti-mouse IgG at 50/~g/ml (Cappel/Cooper Biomedical, Malvern, PA). After 30 min at 4°C, they were washed twice with PBS plus 5 070 FCS and 0.1070 sodium azide and analyzed with an EPICS C Flow cytometer in a manner described below. The panning protocol was successful in depleting the percentage of slg ÷ cells which were identifiable in the unpanned splenocyte population from 22 to 7070 in the panned samples, while the recovered adherent population showed an enrichment in slg + cells of from 22 to 42070. In addition, mitogenesis experiments were performed to further characterize the monoclonal antibody separation protocol. Sample populations of unpanned, non-adherent and adherent splenocytes were cultured in a serum-free medium at 1 x 105 cells/200/xl with either L-15 alone, P H A (2 #g/ml) or LPS (500/~g/ml). In two such tests, the unpanned cells responded to P H A with stimulation indices of 6 and 18 and to LPS with stimulation indices of 5 and 8. The non-adherent populations responded to P H A with stimulation indices of 3 and 14, but were unresponsive to LPS. The recovered adherent population failed to respond to either mito45

gen. The aggressive pipetting required to remove these cells from the surface of the dishes and the binding of slg with its complementary antibody may have affected their capacity to respond. In any case, it became clear that the panning protocol had succeeded in depleting the non-adherent splenocyte population of slg + , LPS sensitive cells.

3.6. Flow cytometric analyses of the anti-IL-2R antibody binding The preparation of the cultured, acid washed, fixed cells and the conditions used for binding FITC (FI*) mouse IgG1, K anti-human IL-2R antibody (Becton-Dickinson, Mt. View, CA) are described in detail elsewhere [5]. Cells from unactivated cultures have been compared to cells activated with P H A with respect to anti-IL-2R binding capacity. Here the Xenopus spleen cells were incubated either without antibody or with 5 nM of the anti-Tac antibody. Prior studies had shown that Xenopus splenocytes would not bind a mouse-anti-DNP IgG1, r antibody unspecifically [5]. The flow cytometric analyses were done with an E P I C S C (Coulter Electronics, Hialeah, FL) using a color compensation circuit board which eliminates background autofluorescence. The percent of fluorescein stained cells was determined as the percentage of Tac + cells that fell to the right of a cursor, preset to eliminate auto fluorescence from consideration. Five thousand cells of each sample were analyzed in each test. The conditions of binding were the same in comparisons of freshly biopsied or in vitro activated cells. 3.7. Competition of rlL-2 and anti-IL-2R antibody

binding In experiments which were designed to test whether the receptors which bind the anti-Tac antibody on toad splenic lymphocytes will also bind h u m a n rlL-2, a competition was effected by prior exposure (30 min at 4 °C) of cells to either a 200- or a 400-fold molar excess of rlL-2. The antibody was then added and the mixture was incubated overnight at 4°C. The percent inhibition is representative of the percent decrease in the 5 nM control antibody binding population as a consequence of prebinding with rlL-2. Assay of anti-Tac antibody binding after prebinding with equivalent concentrations of h u m a n serum albumin (HSA) was used as a test of the specificity of the prebinding with rlL-2. 46

4. Results 4.1. NMU depletion and anti-IL-2R/vlL-2 competi-

tion The alkylating reagent depletes the total cellular population of toad spleens in a very obvious way. As many as 40 adult spleens had to be pooled in order to obtain a large enough cellular population for the several control and experimental groups used in a particular test. The flow cytometric data (Table 1), clearly show that a high proportion of those cells which remain in the spleen 8 days after N M U injection are Tac ÷ . After 3 additional days of culture without P H A in the medium over one-half of these cells (67%, 5407o) will bind the Fl*-anti-IL-2R antibody. However, unlike the situation when undepleted spleen cells are tested [5], the binding of the anti-human-Tac antibody to these cells is not enhanced by in vitro P H A activation (67:61% and 54:51%). The effectiveness of the prebinding with rIL-2 can be assessed by comparison with results obtained after prebinding these splenocytes with the unrelated h u m a n protein, HSA. Since rIL-2 is no more effective than H S A in inhibiting anti-IL-2R antibody binding (61:51% and 51:56%), it is apparent that a 400 × molar excess of rIL-2 fails to specifically inhibit anti-Tac antibody binding when the N M U depleted spleen cell population is tested. Binding data for control groups without Fl~mouse-anti-human IL-2 receptor antibody Table 1 Thebinding of 5 nM Fl*-mouseanti-human IL-2 receptor (R) antibody, after being prebound with PBS or a 400 x molar excess of human rlL-2 or 60/~g/ml of human serum albumin (HSA), to pooled, 3-day cultured spleen cells from 40 adult Xenopus which had been injected 8 days previously with N-CH3-Nnitrosourea (NMU)1. Prebinding protocol

No Ab (negative control) PBS (positive control) rlL-2 HSA

% Cells binding the anti-IL-2R Ab PHA-

PHA

3 67 61 51

3 54 51 56

+

1 NMU is selectivelylymphotoxic in Xenopus. It removes cytotoxic and helper T cell functions. T suppressor function and responses to TNP-Ficoll and TNP-LPS are unaffected.

and one with PBS, rather than rlL-2 or HSA, are also provided. 4.2. The depletion of spleen cells by a mouse

monoclonal anti-Xenopus IgM Ab Freshly biopsied spleen cell populations were tested before and after panning for their capacity to bind Fl*-mouse anti-IL-2R antibody and rlL-2. The flow cytometric data from an experiment comparing an unpanned spleen cell population of 5000 cells, with a similarly sized population of non-adherent (T cell enriched), s l g - , cells, is displayed in Table 2. The anti-Tac binding capacity of the adherent (B cell enriched) cell population had been established in a separate, earlier experiment. The adherent population was not tested for rlL-2 binding, since our data on lectin mitogenesis with these cells, noted earlier in section 3, had suggested that this population may not be fully normal after removal from the panning dishes. Given the considerable expense of these binding competition experiments, which-utilize a 200 to 400× molar excess of rlL-2, we decided that it would still be possible to obtain an understanding of that population by inference from results obtained with the non-adherent population. The data in Table 2 suggest that the proportions of spleen cells which are able to bind anti-Tac anti-

Table 2 The capacity of freshly biopsied unpanned and non-adherent (sIg-) Xenopus splenocytes to bind 5 nM Fl*-mouse antihuman IL-2 receptor antibody and 1.0/zM human rIL-2. The rIL-2 binding capacity is the 070 inhibition of binding by the Fl*-anti-Tac antibodyfollowingpre-binding with a 200-foldmolar excess of rIL-2. Human serum albumin (30 #g/ml of HSA) is used as a control for the prebinding procedure. A cellular pool from three adult spleens was used for this experiment. Prebinding protocol

°70Tac+ cells

070Inhibition

Unpanned splenocytes No anti-Tac Ab Anti-Tac Ab Anti-Tac Ab + rlL-2 Anti-Tac Ab + HSA

<1 72 47 65

35 10

Non-adherent (slg- splenocytes) No anti-Tac Ab Anti-Tac Ab Anti-Tac Ab + rlL-2 Anti-Tac Ab + HSA

1 62 39 53

37 15

body (72:6207o) and rlL-2 (33:37070) are similar when the freshly biopsied untreated population is compared with the non-adherent splenocyte population which had previously been depleted of slg ÷ cells b y a panning procedure with a mouse monoclonal antiXenopus IgM antibody. Although the data are not shown here, it is important also to note that the mean fluorescence per cell of the two populations was the same. Unspecific inhibition of anti-Tac antibody binding (the HSA prebinding data) can account for between 10 and 15070of the blocking effect suggested by the data after prebinding the cells with rlL-2. That is, 22-2507o of the inhibition of anti-Tac antibody binding would seem to be specifically related to the ligand.

5. Discussion The two depletion protocols that we have employed have been used to answer different questions. First, we asked whether N M U would eliminate those splenocytes which are able to express enhanced numbers of IL-2 receptors after lectin (PHA) activation. This is a population of ceils which has already been visualized by flow cytometry [5]. Since rlL-2 fails to stimulate modulation of immune reactivity in this species in the absence of lectin or immunogen activation, these activatable cells would seem likely to be the effectors of the changes in T cell functions that have previously been described [1-4]. In our experiments, the NMU severely depleted the spleens of the injected young adult toads to such an extent that it became necessary to pool the lymphocytes of many spleens (40) in order to perform this test. The data we present here in Table 1 provide a very clear answer to this initial question. The splenocyte population which remained after N M U treatment was not able to respond to P H A by generating a larger proportion of cells with IL-2R. Moreover, when the P H A sensitive population was removed, those remaining cells failed to bind the ligand, rlL-2. We conclude this because prebinding the cells with a 400 x molar excess of rlL-2 did not specifically inhibit binding to the Fl*~anti-Tac antibody. When the HSA was used as an unrelated human protein in the control for the prebinding protocol, it was found to be as ineffective as the lymphokine in this regard. The failure to bind rlL-2 in these tests may be related either to the 3-day 47

culture protocol which was employed after the splenocytes had been recovered from the NMU injected toads or to cellular damage inflicted by the NMU itself. In any case, it seems clear that the NMU sensitive cell population is also the P H A activatable, rlL-2 sensitive population. Thus, the principal effectors of IL-2 dependent immune reactivities in Xenopus would seem to be those lymphocytes which are active in helper T cell regulation of cytotoxic and humoral immune functions. We should note that while NMU will remove cytotoxic T cell function, it is not known whether the NMU eliminates the cytotoxic T effector cells, their associated helper cells or both. The second question that we asked was whether the constitutive Tac ÷ spleen cells, which have been described for this species [5], can be characterized as being either T or B cells. The data presented in Table 2, on the depletion of the spleen cells effected by the monoclonal anti-Xenopus IgM antibody, provide the picture of this population. They show that about two-thirds of the unpanned toad spleen cells will bind the anti-Tac antibody and that about onethird of these will specifically bind rlL-2. The nonadherent spleen population which remains after the slg ÷ cells have been removed binds anti-Tac antibody and rlL-2 to the same extent. Thus, the adherent (slg ÷) population, which also has comparable anti-Tac antibody binding (data not included here), must by inference, also bind r I ~ 2 with equal affinity. If this were not the case, then the proportion of rlL-2 binding cells should have either increased or decreased substantially in the non-adherent cell population. That the mean fluorescence per cell of the two populations tested here was the same is of interest because it tells us that the panning procedure was not selective with respect to whether it removed cells in accordance with the numbers of constitutive IL-2 receptors expressed on their surfaces. Thus, we conclude that both T enriched (slg-) and B enriched (slg +) splenocytes in the toad express constitutive IL-2 receptors and that a similar proportion of them in each population can bind rlL-2. When the percent inhibition of anti-Tac antibody binding which can be attributed to non-specific prebinding with an unrelated human protein (HSA), is subtracted from the percent inhibited by prebinding with the human ligand, the proportion which specifically binds rlL-2 in these experiments is consistent with levels which have been previously reported for other 48

freshly biopsied populations [5]. The data suggest that it would be of interest to turn our attention to rlL-2 modulatory effects on B cell reactivities in this species. Thus far, the only direct effect of rlL-2 on B cells that has been suggested in Xenopus is related to the differentiation of the TNP-specific sub-population (B1), with responsivity to haptenated-immunogenic polysaccharide carriers e.g. Ficoll and Polyvinylpyrrolidone, from its TNP-specific (B2) precursor pool [11, 4]. The evolutionary history of IL-2 function and, in particular, its relationship to the regulation of B cell activities would seem to be of considerable import since antibody producing cells are present in even the most primitive of the extant vertebrates, the cyclostomes [12]. These jawless creatures e.g., the hagfish and the lamprey, do not have thymuses as organized lymphoid structures.

Acknowledgements We are grateful for the partial support of this research by grants AI-12846 (to L.N.R.), RR 07168 (to Reed College) and RR 00163 (to the Oregon Regional Primate Research Center) from the National Institutes of Health, Bethesda, MD.

References [1] Ruben, L. N., Barr, K., Clothier, R. H., Nobis, C. and Balls, M. (1985) Dev. Comp. Immunol. 9, 811. [2] Ruben, L. N., Clothier, R. H. and Balls, M. (1985) Cell. Immunol. 93, 299. [3] Ruben, L. N. (1986) Immunol. Lett. 13, 227. [4] Ruben, L. N., Clothier, R. H., Mirchandani, M., Wood, P. and Balls, M. Immunol. 61, (in press). [5] Langeberg, L., Ruben, L. N., Malley, A., Shiigii, S. and Beadling, C. (1987) Immunol. Lett. 14, 103. [6] Piantelli, M., Larocca, L. M., Aiello, F. B., Maggiano, N., Carbone, A., Ranelletti, E O. and Musiani, P. (1986) J. Immunol. 136, 3204. [7] Mukaida, N., Kasahara, T., Hosoi, J., Shiori-Nakano, K. and Kawai, T. (1986) Immunol. 57, 137. [8] Waldmann, T. A. (1986) Science 232, 727. [9] Balls, M., Clothier, R. H., Hodgson, R. and Berridge, D. (1980) in: Development and Differentiation of Vertebrate Lymphocytes (J. D. Horton, ed.) pp. 183-194, NorthHolland, Amsterdam. [i0] Fazekas de St. Groth, S. and Scheidegger, D. (1980) J. Immunol. Methods 35, 1. [11] Ruben, L. N., Clothier, R. H. and Balls, M. (1986) Thymus 8, 341. [12] Ruben, L. N. and Edwards, B. E (1978) Dev. Comp. Immunol. 2, 753.