Immunology 1979 36 527

Functional maturation of neonatal spleen cells

RITA BOSING-SCHNEIDER University of Konstanz, Department of Biology, Immunology Unit, Konstanz, Germany

Received 9 March 1978; acceptedfor publication 14 July 1978

functional T and B cells in a newborn animal. In order to avoid complications due to simultaneous development of different cell types, T and B cells of the spleen were studied under separate culture conditions. The ontogenetic maturation of T lymphocytes was measured by the ability to produce a T-cell replacing factor (TRF) which can restore a T-cell deficient system; the maturation stage of splenic B cells was judged by their capacity to accept the T-cell signal in the form of a T-cell replacing factor and to differentiate, giving rise to antibody-producing cells. In the culture method the developmental stage was defined by the time of explantation. Since the duration of the culture was short (4 days) further ontogenetic differentiation in vitro was not expected to interfere. Therefore possible uncontrolled helper activities of the cellular environment are reduced in this system as compared to irradiated hosts.

Summary. Maturation of neonatal spleen cells was studied in vitro with a cell population restricted with respect to functional properties. It is shown that the onset of the immune response to SRBc in post-natal mice was delayed because B and T cells were incompe;. tent. It appears, however, that the development of these two cell populations does not occur in parallel. Since the addition of adult macrophages failed to overcome the incompetence of neonatal B cells in the presence of a T cell replacing factor, it is suggested that the late appearance of immune competence is due to the inability of B cells to process a T-cell signal. INTRODUCTION

Although considerable progress has been made in recent years in defining stages of lymphocyte differentiation with surface markers, any approach to the functional maturation stage of a particular lymphocyte population by adoptive transfer experiments with precursor cells is complicated by the possible contribution of the irradiated animal. Therefore a tissue culture system with a restricted cell population is preferable for studying the delayed development of immune function in post-natal mice. This paper is concerned with the differentiation of Correspondence: Dr Rita B6sing-Schneider, Universitat Konstanz, Fachbereich Biologie, Postfach 7733, D-775

MATERIALS AND METHODS

Animals BALB/c nu/nu mice obtained from Bomholtgard. Denmark, were used as a source of neonatal and adult spleen cells deficient in T cells. Donors for normal spleen cells were either BALB/c or BALB-Igb of our own breeding stock. NMRI-mice were purchased from Tierzuchtanstalt Hannover, Germany. TRF

Konstanz, Germany

T-cell replacing factor was produced in spleen cell cultures of normal mice with different genetic back-

0019-2805/79/0300-0527$02.00 ©) 1979 Blackwell Scientific Publications 527

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grounds after stimulation by concanavalin A (2 ug/ml); it was added either to nude cell cultures according to the method of Schimpl & Wecker (1972), or to normal neonatal spleen cells deficient in immunocompetent cells, for antibody production in vitro.

Cell culture Spleen cells from neonatal or adult mice were prepared and cultured according to the method of Mishell & Dutton (1967) and incubated in the presence of 2-mercapto-ethanol for 4 days (Click, Benck & Alter, 1972). Sheep red blood cells (SRBC) were used as antigen at a dose of 5 x 106 per I07 cultured cells. After 4 days of in vitro incubation in the presence or absence of TRF, four culture dishes of each group were pooled and the number of antibody-producing cells was determined in triplicate with a modified Jerne plaque technique. Preparation of macrophages Spleen cells (1 -5-3-0 x 107 cells/ml) from adult mice were incubated in Eagle's medium supplement with 10% foetal calf serum. After 3 h, non-adherent cells were removed. The remaining adherent cells were washed twice with Eagle's medium and used as a source of enriched functional macrophages. The number of cells washed off the adherent layer was counted, and the number of attached cells was calculated by difference. Under these conditions the number of attached cells varied between 9 x 105 and 1 x 106 cells per culture dish after seeding of 15 x 107 spleen cells/ml. The number of attached cells increased to 2 x 106 cells per dish after seeding of 3 x 107 spleen cells. Both concentrations of adherent cells were able to restore an in vitro anti-SRBC response in a nonadherent population. 1-5 x 107 neonatal spleen cells either from normal BALB/c or nude mice, were layered over these adherent cells and stimulated with SRBC in the presence or absence of TRF.

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1-2 3-4 5-6 7-8 9-10 11-Z2 17-1821-22 60-70 60-70 Age of spleen cell donors for the production of TRF (days)

Figure 1. Functional maturation of T cells. Spleen cells from adult BALB/c-nude mice were cultured in the presence of antigen with the addition of a supernatant derived from either neonatal or adult congeneic spleen cells (BALB-Igb), stimulated by Con A. After 4 days the number of plaqueforming cells was counted as an indication of T-cell replacing activity of the various supernatants used (open columns). Control cultures were incubated in the presence of antigen but in the absence of a T-cell replacing supernatant (hatched column). Bars indicate the standard deviations.

shown that supernatants collected 24 h after stimulation of adult (60-70 days) spleen cells can replace T cells in cultures from adult nude mice and induce antibody production in the presence of SRBC. Cultures of spleen cells from adult nude mice yield ten times as many PFC in the presence of TRF as in its absence. In order to show this effect, TRF has to be obtained from cultures of adult donors. Therefore the magnitude of the immune response in T-cell deficient cultures indicates the strength of a T-cell replacing supernatant obtained from postnatal spleen cells. As we can see in Fig. 1, supernatants from neonatal (1-2 days) spleen cells did not contain T-cell replacing activity, but mice at the age of 1 or 2 weeks had sufficient numbers of mature T cells in their spleens to produce a T-cell replacing factor. In Fig. 2, experiments are summarized which show that there was no time difference in the development of cells able to produce TRF when allogeneic C57B1 ten mice were used instead of congeneic BALB-Igb mice. Here also in the second week after birth, spleen cells differentiated to produce helper activity for the humoral immune response in T-cell deficient cultures.

RESULTS

(1) Ontogeny of T-cell function Nude spleen cell cultures were stimulated with SRBC and used as a defined B-cell population, deficient in functional T cells. In order to study the stage of T-cell maturation in neonatal mice, supernatants of cultured cells, explanted from mice at different ages, were examined for their T-cell replacing capacity. In Fig. 1 it is

(2) Ontogeny of B-cell function To investigate whether the inability of normal spleen cells from newborn animals to respond to SRBC is due only to the absence of functional T cells or whether the B-cell population is also immature, the following experiments were carried out. (a) Spleen cells from newborn mice of different ages

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Maturation of neonatal spleen cells

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60-70 7-18 9-10 11-12 1-2 3-4 5-6 Age of spleen cell donors for the production of TRF (days)

Figure 2. Functional maturation of T cells allogeneic to the nude spleen cell culture. Adult nude spleen cells were incubated in the presence of antigen with the addition of a supernatant derived from allogeneic neonatal (C57B1 10) spleen cells. T-cell replacing activity is indicated by the magnitude of PFC response obtained after 4 days in cultures stimulated with antigen. Bars indicate the standard deviations.

were cultured in the presence of antigen and of TRF with strong T-cell replacing capacity, derived from mature cells. After 4 days the magnitude of the immune response in cultures derived from newborn mice was compared with cultures from older mice. The results are shown in Fig. 3. Unlike the control cultures with cells from adult mice, the addition of TRF to neonatal spleen cells did not lead to an increase in the number of plaque-forming cells after 4 days of culture. As one can see in Fig. 3, large numbers of antibody-

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producing cells were not obtained in cultures prepared from mice less than 2 weeks old. Cells which can be converted to plasma cells in the presence of TRF seem to appear later than mature T cells in the same spleen cell population (compare Figs I and 2). (b) Since suppressor T cells might also occur in the normal neonatal spleen, cells from very young T-cell deficient animals (nude mice) were also examined for the ability to respond in vitro to SRBC in the presence of TRF. The data from these experiments are summarized in Fig. 4. Spleen cells from newborn nude mice do

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3-4 5-6 8-9 11-12 15-16 25-26 Age of spleen cell donors (days)

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Figure 4. Ontogeny of B-cell function in T-cell deficient cultures. Neonatal spleen cells derived from BALB/c nude mice were incubated in the presence (open columns) or absence (hatched column) of a powerful T-cell replacing factor for 4 days. Shown is the average of three experiments. Bars indicate the standard deviations.

not contain appreciable numbers of mature cells which can respond in vitro in the presence of a powerful TRF. They do not differ from normal neonatal cells in their inability to accept the T-cell signal for differentiation into plasma cells.

(3) Function of adult macrophages in neonatal cell cultures

3-4

5-6

8-9

11-12

60-70

Age of spleen cel donors (days)

Figure 3. Ontogeny of B-cell function in the presence of a T-cell replacing factor (TRF). Neonatal spleen cells derived from BALB-Igb donors were stimulated with antigen in the presence (open columns) or absence (hatched columns) of TRF. After 4 days the number of antibody producing cells was determined. Shown is the average of four experiments. Bars indicate the standard deviations.

Since it has been reported that peritoneal macrophages from newborn mice differ morphologically (Hardy, Globerson & Danon, 1976) from those obtained from adult mice, experiments were carried out to investigate whether the delayed onset of immune induction in neonates was due to immaturity of splenic macrophages. Neonatal spleen cells were added to different numbers of adult adherent cells and incubated in the presence ofTRF and antigen. After 4 days the number of antibody-producing cells in these cultures was determined and compared to the number

Rita Bosing-Schneider

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F c E D A B Figure 5. Failure of adult ma crophages to convert neonatal spleen cells into a mature cell population in the presence of TRF. Spleen cells derived frorm donors of different ages were incubated in vitro. After 4 dayss the number of plaque-forming cells indicates whetherculture sare mature or incompetent for immune induction. A, adult s adult spleen cells in the presenice of antigen; C, neonatal (67 days) spleen cells stimulated iwith antigen; D, neonatal (6-7 days) spleen cells stimulated iwith antigen in the presence of TRF; E, neonatal spleen cellIs stimulated with antigen and TRF in the presence of aduilt macrophages (derived from 1-5 x I07 adult spleen cells/ml)) F, neonatal spleen cells stimulated with antigen and TRF iin the presence of adult macrophages (derived from 3 x I0" adult spleen cells/ml).

of plaque-forming cells olbtained from cultures without additional macrophag;es. The data in Fig. 5 show that the addition of adulIt macrophages to neonatal cells did not lead to a consvincing increase in antibody production in the presenIce of TRF. The slight enhancement may be due tto contamination by a few adult lymphocytes left orn the layer of adult macrophages after the non-adhe rent cells were washed off.

DISCIUSSION Extensive studies have been made on surface characteristics of lymphocytes , during ontogeny (Spear, Wang, Rutishauser & Ed(elman, 1973; Nossal & Pike, 1973; Gelfand, Asofsky BE Paul, 1974a; Scher, Scharrow, Wistar, Asofsky &'William, 1976). Since B-cell activity has been detected even in the yolk sac or in the foetal liver (Nossal & Pikee, 1973; Tyan & Herzenberg,

1968; Chiscon & Golub, 1972; Umiel & Globerson, 1974; Melchers, 1977; Raff, Megson, Owen & Cooper, 1976) the delayed onset of the immune response after birth is often attributed to the absence of mature T cells or macrophages in newborn animals (Spear et al., 1973; Spear & Edelman, 1974; Hardy et al., 1973). Because of the fact that 95% of thymus cells and 20% of spleen cells from newborn mice carry the 0-antigen (Spear et al., 1973; Stobo & Paul, 1972), the inability of neonatal cells to respond to different antigens might be due to lack or suppression of T-cell function, rather than to T-cell absence. The results presented here indicate that T- and B-cell maturation may not be complete at the same point in time. As measured by production of TRF, the data have shown that splenic T cells are immature during the neonatal stage but may become functional earlier than B cells. The capability of neonatal spleen cells to produce TRF seems to be related to the magnitude of mitogen responsiveness of thymocytes in newborn mice (Stobo & Paul, 1972; Mosier, 1974). Because it has been shown that differentiation and function ofT cells in particular are influenced by cyclic AMP (Bosing-Schneider, 1975; Bosing-Schneider & Haug, 1976a, b; Singh & Owen, 1975; Scheid, Hoff-

Hammerling, Abborr, Boyse, Cohen, Hooper, Schulof & Goldstem, 1973) one must investigate whether the starting signal for differentiation to TRF production is missing in the neonatal period.

man, Komuro,

Since It has been reported that Ig-bearing as well as

antigen-recognizing cells can be detected in the neonatal spleen in appreciable numbers (Gelfand, Elfenbein, Frank & Paul, 1974b; Rosenberg & Parish,

1977), one can postulate that the receptor for antigen

recognition has already been developed on neonatal B cells, but the acceptor site for the T-cell signal might be missing. Based on these results we chose an antigen dose for neonatal cells in the same range as for adult cells. Because of the fact that neonatal spleen cells from nude mice cannot be stimulated in vitro in the presence of TRF we conclude that this unresponsiveness is not caused by neonatal suppressor cells. This corresponds also to other experiments which showed that neonatal spleen cells cannot prevent adult lymphocytes from being stimulated in vitro (R. B6sing-Schneider, unpublished results). But since adult nude spleen cells treated with thymus-derived lymphocytes, exhibited a similar unresponsiveness in vitro as neonatal cells (BosingSchneider & Haug, 1976b) one can still question whether B cells in the neonatal stage might be more

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Maturation of neonatal spleen cells

accessible to suppression in vitro than adult lymphocytes. As it is known that macrophages from neonatal mice are functionally immature (Hardy et al., 1973), the question may arise whether the immaturity of these cells is responsible for the delayed onset of the immune induction in vitro, because macrophages from newborn mice seem to differ from cells of adult mice not only morphologically but also with respect to surface charge (Hardy, Skutelsky, Globerson & Danon, 1976). The present experiments demonstrate that adult macrophages fail to convert neonatal spleen cells to a functionally mature cell population. These results are not at variance with experimental data of Rosenberg & Cunningham (1975) who showed an increase of plaque-forming cells in cultures from neonatal mice, since they simulated these cells in the presence of a strong B-cell mitogen (LPS). This is known to activate polyclonally even foetal liver cells to Ig production (Melchers, 1977). Similar data were obtained by Stocker who found that neonatal spleen cell cultures could be stimulated to antibody production only if these cells were matured previously by pre-culturing for 3 days (Stocker, 1977). Based on the hypotheses of Dukor & Hartmann (1973) and of Parish (1975) that complement plays a key role during immune induction, one might suggest that the late appearance of complement receptors (Gelfand, et al., 1974b; Rosenberg & Parish, 1977) on B cells is related to the inefficiency of TRF in giving an inductive signal to B cells from newborn animals. This would correspond to the data and the model of Pepys (1974) who postulates that complement plays an important role in T- and B-cell co-operation. The delayed onset of the in vitro immune response against SRBC in neonatal cells is in accordance with the onset of the IgG immunoglobulin production in non-immunized normal or nude mice which starts in the third week of age and reaches reasonable titres about the fourth week after birth (Kolb, Di Pauli & Weiler, 1974; 1976). If one compares in vivo (Playfair, 1968) and in vitro experiments (Fidler, Chicson & Golub, 1972 and Spear & Edelman, 1974) concerned with the development of antibody production in neonatal BALB/c mice, it appears that the onset of the immune response against SRBC is delayed in vitro. This difference might reflect a different maturity of the spleen cell population in comparison with other lymphoid organs. The present experiments, which deal with different functional compartments within the neonatal spleen,

may help to elucidate those reports in which the in vitro response of neonatal cells is markedly reduced up to the fourth week after birth compared to cultures from adult donors.

ACKNOWLEDGMENTS I thank Prof. Dr E. Weiler and Dr I. J. Weiler for reading the manuscript and stimulating discussions. The excellent technical assistance of Mrs M. Haug is acknowledged.

REFERENCES BOSING-SCHNEIDER R. (1975) Differential effects of cyclic AMP on the in vitro induction of antibody synthesis. Nature (Lond.), 256, 137. BOSING-SCHNEIDER R. & HAUG M. (1976a) Role of cyclic AMP on differentiation of T and B lymphocytes during the immune induction. Cell Immunol. 27, 121. BOSING-SCHNEIDER R. & HAUG M. (1976b) Induction and abrogation of unresponsiveness in nude mouse cells. J. exp. Med. 144, 1458. CHISCON M.O. & GOLUB E.S. (1972) Functional development of the interacting cells in the immune response. J. Immunol. 108, 1379. CLICK R.E., BENCK L. & ALTER B.J. (1972) Enhancement of antibody synthesis in vitro by mercaptoethanol. Cell Immunol. 3, 156. DUKOR P. & HARTMANN K.-U. (1973) Hypothesis. Bound C3 as the second signal for B-cell activation. Cell Immunol. 7, 349. FIDLER J.M., CHISCON M.O. & GOLUB E.S. (1972) Functional development of the interacting cells in the immune response. J. Immunol. 109, 136. GELFAND M., ASOFSKY R. & PAUL W.E. (1974a) Ontogeny of B lymphocytes. Cell Immunol. 14, 460. GELFAND M.C., ELFENBEIN G.J. FRANK M.M. & PAUL W.E. (1974b) Ontogeny of B lymphocytes. II. Relative rates of appearance of lymphocytes bearing surface immunoglobulin and complement receptor. J. exp. Med. 139, 1125. HARDY B., GLOBERSON A. & DANON D. (1973) Ontogenetic development of the reactivity of macrophages to antigenic stimulation. Cell Immunol. 9, 282. HARDY B., SKUTELSKY E., GLOBERsoNA. & DANON D. (1976) Ultrastructural differences between macrophages of newborn and adult mice. J. Reticul. Soc. 19, 291. KOLB C., DI PAULI R. & WEILER E. (1974) Induction of IgG by lipid A in the newborn mouse. J. exp. Med. 139,467. KOLB C., DI PAULI R. & WEILER, E. (1976) Induction of IgG in young nude mice by lipid A or thymus grafts. J. exp. Med. 144, 1031. MELCHERS F. (1977) B lymphocytes development in fetal liver. I. Development of reactibilities to B-cell mitogens in vivo and in vitro. Europ. J. Immunol. 7, 476. MISHELL R.J. & DUTTON R.W. (1967) Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med. 126,423.

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MOSIER D. (1974) Ontogeny of mouse lymphocyte function. I. Paradoxical elevation of reactivity to allogeneic cells and phytohemagglutin in BALB/c fetal thymocytes. J. Immunol. 112, 305. NOSSAL G.J.V. & PIKE B.L. (1973) Studies on the differentiation of B lymphocytes in the mouse. Immunology, 25, 33. PARISH C.R. (1975) Separation and functional analysis of subpopulations of lymphocytes bearing complement and Fc receptors. Transpl. Rev. 25, 98. PEPYS M.B. (1974) Role of complement in induction of antibody Production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymusindependent antibody response. J. exp. Med. 140, 126. PLAYFAIR J.H.L. (1968) Strain differences in the immune response of mice. I. The neonatal response to sheep red cells. Immunology, 15, 35. RAFF M.C., MEGSON M., OWEN J.J.T. & COOPER M.D. (1976) Early production of intracellular IgM by B-Lymphocyte precursors in mouse. Nature (Lond.), 259, 224. ROSENBERG Y.J. & CUNNINGHAM A.J. (1975) Ontogeny of the antibody-forming cell line in mice. I. Kinetics of appearance of mature B cells. Europ. J. Immunol. 5, 444. ROSENBERG Y.J. & PARISH C.R. (1977) Ontogeny of the antibody-forming cell line in mice. IV. Appearance of cells bearing Fc-receptors, complement receptors, and surface immunoglobulin. J. Immunol. 118, 612. SCHEID M.P., HOFFMANN M.K., KOMURO K., HAMMERLING U., ABBORR J., BOYSE E.A., COHEN G.H., HOOPER J.A., SCHULOF R.S. & GOLDSTEIN A.L. (1973) Differentiation

of T cells induced by preparations from thymus and by nonthymic agents. J. exp. Med. 138, 1027. SCHER I., SCHARROW S.O., WISTAR R., ASOFSKY R. & WILLIAM E.P. (1976) B lymphocytes heterogeneity: ontogenetic development and organ distribution of B-lymphocyte population defined by their density of surface immunoglobulin. J. exp. Med. 144,494. SCHIMPL A. & WECKER E. (1972) Stimulation of IgG antibody response in vitro by T-cell replacing factor. J. exp. Med. 137, 547. SINGH U. & OWEN J.J.T. (1975) Studies on the effect of various agents on the maturation of thymus stem cells. Europ. J. Immunol. 5, 286. SPEAR P.G., WANG A., RUTISHAUSER U. & EDELMAN G.M. (1973) Characterization of splenic lymphoid cells in fetal and newborn mice. J. exp. Med. 138, 557. SPEAR P.G. & EDELMAN G.M. (1974) Maturation of the humoral immune response in mice. J. exp. Med. 139,249. STOBO J.D. & PAUL W.E. (1972) Functional heterogeneity of murine lymphoid cells. II. Acquisition of mitogen responsiveness and of o-antigen during the ontogeny of thymocytes and 'T' lymphocytes. Cell Immunol. 4, 367. STOCKER J.W. (1977) Functional maturation of B cells in vitro. Immunology 32, 275. TYAN M.L. & HERZENBERG L.A. (1968) Studies on the ontogeny of the mouse immune system. J. Immunol. 101, 446. UMIEL T. & GLOBERSON A. (1974) Analysis of lymphoid cell types developing in mouse fetal liver. Differentiation, 2, 169.

Functional maturation of neonatal spleen cells.

Immunology 1979 36 527 Functional maturation of neonatal spleen cells RITA BOSING-SCHNEIDER University of Konstanz, Department of Biology, Immunolog...
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