A Factor That Can be Used to Regulate an In Vitro Primary Immune Response (mice/rauscher leukemia virus/spleen

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 69, No. 3, pp , March 1972 A Factor That Can be Used to Regulate an In Vitro Primary Immune Response (mice/rauscher leukemia virus/spleen JAMES WATSON AND MARILYN THOMAN cells/plaque-forming cells) The Salk Institute for Biological Studies, Post Office Box 1809, San Diego, California Communicated by Robert W. Holley, December 20, 1971 ABSTRACT A factor has been isolated that supports the stimulation of an immune response against heterologous erythrocyte antigens in mouse spleen cells cultured in a medium that contains deficient serum. The factor is secreted by certain spleen cells that are obtained in permanent culture after leukemia virus infection. The factor permits antigen-sensitive cells derived from bone marrow to mature to antibody-forming cells. This factor can be used in controlling the initiation of a primary immune response in vitro. Cultures of mouse spleen cells contain all the cell types required for the initiation of a primary immune response to heterologous erythrocyte antigens (1). Lymphocytes derived from bone marrow have been identified as the direct progenitors of the antibody-forming cells (2); lymphocyte differentiation depends upon the specific stimulation resulting from interaction with antigens in collaboration with other cell types (3,4). In this paper we describe a new method of controlling the initiation of an in vitro primary immune response. Primary immune responses are elicited against erythrocyte antigens in mouse spleen cultures only when the medium is supplemented with select batches of fetal bovine serum (FBS). Many batches of FBS, here termed deficient, do not sustain this response (1). A factor has been isolated here that supports the stimulation of primary immune responses against erythrocyte antigens in cultures supplemented with deficient FBS. The source of the factor is culture supernatants of several cell lines established by infection of spleen cultures with a murine leukemia virus. The experimental conditions for the initiation of an in vitro primary immune response in the deficient culture medium are analyzed here. Elsewhere we consider experiments that suggest that the factor may permit the expression of a specific cell type required for the in vitro response (Watson, J. D. & Prichard, J., submitted for publication). MATERIALS AND METHODS Spleen Cell Cultures. We followed exactly the procedures described elsewhere for the induction of in vitro immune responses to heterologous erythrocyte antigens in suspensions of mouse spleen cells (1). Primary immune responses were studied in suspensions of spleen cells from 6- to 8-week old unimmunized C57BL/6 mice (Jackson Laboratories). Secondary immune responses were investigated in mice immunized Abbreviations: PFC, plaque-forming cells; FBS, fetal bovine serum. intravenously with 0.1 ml of a 10% solution of sheep or horse 3 days before culture. Each culture contained 1.2 X 107 spleen cells in 1 ml of Eagle's minimum essential medium containing 5% FBS and 2- or horse (Colorado Serum Co.). At various days after culturing, the number of direct hemolytic plaque-forming cells (PFC) in each culture was determined by a microscope slide assay (1). All results are expressed as the number of PFC per culture (1.2 X 107 input cells), averaged from triplicate cultures. Cell recoveries after 4-5 days were in the range 20-35% of the input cell number. The background number of PFC obtained in control cultures lacking heterologous erythrocyte antigens was determined for each experiment ( PFC per input of 1.2 X 107 cells); these numbers have been subtracted from the reported data. Cultures were fed daily with a nutritional mixture (1) supplemented with the FBS used in preparation of the culture. Fetal Bovine Serum. Batches of FBS were screened for their ability to support in vitro primary immune responses. Serum that supports a primary immune response in vitro has been designated normal FBS. The batches of normal FBS used are E and E obtained from Reheis Chemical Corp. A number of batches did not support an in vitro primary immune response and have been designated deficient. The deficient FBS chosen for these studies was found to be deficient by Dr. R. I. Mishell; it is batch no. E20102 from Reheis. Cell Lines. ML-1 and ML-2 cell cultures were established from spleen cell cultures from BALB/c mice that were infected with Rauscher leukemia virus. Spleen cultures from mice immunized with sheep 3 days earlier were prepared. After 3 days in culture, the cells were infected with Rauscher leukemia virus (5). Virus-infected ML-1 and ML-2 cell lines grow as attached cultures in Eagle's medium supplemented with either 10% normal or deficient FBS; both cultures have now been passaged 16- to 20-times in the absence of sheep. ML1 and ML2 are mixed cell cultures. JLS-V5 cells were derived originally by infection of a mixed culture of spleen and thymus cells from BALB/c mice with Rauscher leukemia virus (6). A clone of JLS-V5 and of 3T3- NIH cells was isolated and grown (7). 3T3-RLV is a cell line obtained from 3T3-NIH infected with Rauscher leukemia virus; this cell line continues to secrete virus. Mouse myelomas and lymphomas were derived from oil-induced tumors of BALB/c mice (8). All myeloma and lymphoma cells used here were infected with Gross-type leukemia viruses (9). Preparation of Factor. Monolayers of ML-1, ML-2, or JLS-V5 cells were grown in Eagle's medium containing 10% 594

2 Proc. Nat. Acad. Sci. USA 69 (1972) deficient FBS, in roller bottles. When cultures reached confluency, the cells were washed twice with Eagle's medium without serum and then incubated for 48 hr in Eagle's medium without serum at a density of about 106 cells/ml. The medium was then removed and centrifuged at 2000 X g for 10 min to remove any cells and at 100,000 X g for 90 min to remove leukemia virus particles. The factor was precipitated from the medium with 50% ammonium sulphate. The precipitate was collected by centrifugation at 12,000 X g for 10 min, dissolved in 0.01 M Tris.HCl (ph 7.4), and dialyzed against the same buffer; the insoluble precipitate was removed by centrifugation and discarded. Soluble material was fractionated by Sephadex G-100 column chromatography with 0.05 M Trist HCO (ph 7.4) as the eluant buffer. Column fractions were collected, sterilized by filtration through 0.45-,Am Millipore filters, and stored at -20. Protein concentrations were determined (10), and the factor was prepared from other cell lines as described in the legend for Table 2. RESULTS Effect of serum on spleen cell cultures The experiments described in Fig. 1 show the differences between primary and secondary immune responses elicited against sheep in mouse spleen cultures supplemented with either normal or deficient FBS. When cultures are prepared from spleens of unimmunized mice in medium supplemented with normal FBS, an exponential increase in the number of PFC from day 3 to day 5 represents a primary immune response (Fig. 1A). However, when identical cultures are prepared in a medium supplemented with deficient FBS, the number of PFC does not increase. Similar results are obtained with horse or burro as antigens (data not shown). In comparison, cultures prepared from mice that have been immunized with sheep 3 days previously show a similar secondary response in vitro in media supplemented with normal or deficient FBS (Fig. 1B). The only difference between in vitro secondary responses is observed at the start of the response, the number of PFC in cultures supplemented with deficient FBS is never as high as in identical cultures 10 A B DDA a.' FIG. 1. A comparison between primary and secondary immune responses elicited against sheep in cultures containing either 5% (A or 0), or deficient (A or 0) FBS. (A) Primary response of spleen cells from unimmunized mice. (B) Secondary response of spleen cells from mice immunized 3 days earlier. No difference was seen in the recovery of cells from cultures containing normal or deficient FBS in cultures eliciting either primary or secondary responses. Regulation of In Vitro Immune Responses 595 w -J W. C.) IL. 105 cat A& INA 14 - A* I 1 3., HOURS WITHOUT ERYTHROCYTES FIG. 2. Survival of spleen cell types in culture. Spleen cells from unimmunized mice were incubated in medium containing normal FBS (A-A) or deficient FBS (A-A) without sheep and, at the times indicated, transferred to fresh medium containing normal FBS and sheep. The PFC assays were performed 4 days after the initial cultures -were established, i.e., 3 days after the addition of to the transferred cultures. Cell recoveries from cultures containing normal or deficient FBS at each time indicated were identical. supplemented with normal FBS. By days 4 and 5 in culture, the responses become identical (Fig. 1B). Thus, in vitro primary immune responses are not induced in spleen cell cultures in media supplemented with deficient FBS, whereas in vitro secondary responses are induced in media supplemented with deficient FBS. The experiment described in Fig. 2 compares the survival of limiting spleen cells required for the production of in vitro primary responses in culture media supplemented either with normal or with deficient FBS. When spleen cells from unimmunized mice are incubated at 37 in medium with normal or deficient FBS for up to 30 hr in the absence of sheep and then transferred to fresh culture medium supplemented with normal FBS and, the primary immune responses that are then elicited are identical (Fig. 2). Note that cultures incubated hr without sheep and then exposed to the, give a response on day 4 (or 3 days after addition of the ) (Fig. 2) similar to that elicited by cells exposed 3 days from the start to erythrocyte antigens (Fig. 1A). The conclusion is that deficient FBS does not support the differentiation of one or more of the cell types involved in the in vitro immune response since its effects are reversible, but it maintains these cells in a viable form (Fig. 2). Stimulation of primary immune responses in vitro in deficient culture media The data presented in Fig. 3 show that when tissue culture medium without serum is conditioned by incubation for 24 hr with leukemia virus-infected cell lines JLS-V5, MI-i, or ML-2, the medium will stimulate an in vitro primary immune response in spleen cell cultures supplemented with deficient FBS. All stimulatory activity is recovered from the culture supernatant by precipitation with 50% ammonium sulfate. Fractionation of the material collected in the ammonium sulfate precipitate by Sephadex G-100 gel filtration at ph 7.4 results in the separation of two distinct components (Fig. 4A, I and II). When each component is assayed for

3 596 Immunology: Watson and Thoman Ir -J D L PERCENT [v/vl CONDITIONED MEDIUM FIG. 3. Effect of conditioned medium on in vitro primary immune responses. Cultures containing 107 JLS-V5 (A), ML-1 (-), or ML-2 (N) cells were incubated in Eagle's medium without FBS for 24 hr. The medium was then removed, filtered through a 0.45-,m Millipore filter and diluted with fresh medium containing 5% deficient FBS (final concentration) in the range indicated. The ability of the conditioned medium to support the stimulation of primary responses in vitro in spleen cultures was then assayed on day four. stimulatory activity over a wide range of protein concentrations, most of the activity is found associated with component I (Fig. 4B). By use of immunoglobulin protein subunits as markers, the molecular weight of component I was found to be about 100,000 (data not shown). The kinetics of induction of the primary immune response in cultures containing deficient FBS supplemented with various concentrations of component I are described in Fig. 5A. With 25 and 100Mug of component I per culture, the I 0.6 z t min < 0.2 A I H. '''' IIX~ S. I_.', FRACTION NUMBER, U" / -,,, A/ I E. I., I * II - A,,, av, 1'. 10 B N-FBS s &-^& C, / OX 103 I~ X. a jlg PROTEIN FIG. 4. (A) Gel filtration of JLS-V5 factor activity. A column (90 X 3 cm) containing Sephadex G-100 was equilibrated in 0.05 M Tris HCl (ph 7.4) buffer at 4. The 50% ammonium sulfate precipitate obtained from 48-hr conditioned medium with JLS-V5 cells (Methods) was layered on the column; 4-ml fractions were collected; 0.05 M Tris HCl (ph 7.4) was used as the eluant. The fractions indicated as components I and II were pooled, dialyzed against water, lyophilized, and suspended at equal protein concentrations. (B) The activity of components I (A) and II (0) have been assayed; various protein concentrations in spleen cell suspensions from unimmunized mice were used. The culture medium was supplemented with 5% deficient FBS, and triplicate cultures were assayed with the indicated concentrations of components I and II. The average PFC per culture at day 4 has been plotted. Note the PFC response in control cultures with normal (N) FBS (Methods). immune response in vitro is virtually identical with the response elicited against sheep in cultures supplemented with normal FBS. As the concentration of component I-in each culture is decreased, the lag period before the proliferation of PFC is detected increases; the number of PFC at the peak of the in vitro response decreases, but the rate of appearance of PFC during the proliferative phase remains constant for all concentrations of component I added (Fig. 5A). Component I is required to be present continuously for stimulatory activity. The rate of appearance of PFC decreases when spleen cells are transferred to medium containing deficient serum in the absence of component I (Fig. 5B). TABLE 1. Proc. Nat. Acad. Sci. USA 69 (1972) Specificity of factor secreted from JLS-V5 cells PFC/culture Coin- assayed on ponent Sheep Horse I eryth- eryth- Components of cultures (jsg) rocytes rocytes (A) 1.2 X 107 unimmunized spleen cells with 5% normal FBS and (B) 1.2 X 107 unimmunized spleen cells with 5% 50 5, deficient FBS and 100 4, * 410 NT 500* 560 NT 1000* 370 NT (C) 1.2 X 107 unimmunized spleen cells with 5% deficient FBS and X 106 horse (D) 1.2 X 107 immunized - 59, spleen cells with 5% 50 63, normal FBS and , (E) 1.2 X 107 unimmunized 50 0 NT spleen cells in medium NT without FBS, with NT (F) 1.2 X 107 immunized 52,300 NT spleen cells with 5% 50 50,480 NT deficient FBS and ,300 NT Immunized spleen cells were taken from mice injected intraperitoneally with sheep 3 days before culture. Component I was prepared from JLS-V5 medium as described in the legend to Fig. 4A. * Material similar in size to component I isolated from media with deficient FBS. About 100 mg of deficient FBS protein was used as the starting material; the 50% ammonium sulfate precipitate was fractionated by Sephadex G-100 column chromatography, and the fractions that correspond to the same region as component I of JLS-V5 (Fig. 4A) were collected and concentrated. The PFC per culture were assayed at day four with sheep or horse cells in the assay. NT-not tested.

4 Proc. Nat. Acad. Sci. USA 69 (1972) The data presented in Table 1 include some of the specific properties of component I. These are: (i) The in vitro primary response is antigen-dependent (Table 1A, B, and C). Component I does not nonspecifically stimulate the cultures in the absence of antigen. (ii) In vitro primary or secondary immune responses elicited in spleen cell cultures supplemented with normal FBS are not markedly stimulated by component I (Table IA and D). The factor restores a lost activity only. (iii) Component I does not substitute for FBS in stimulating a primary immune response in vitro (Table IE). (iv) In vitro secondary immune responses in spleen cell cultures supplemented with deficient FBS are not affected by the addition of component I (Table IF). (v) The factor from ML-1, ML-2, or JLS-V5 cell supernatants does not appear to be a contaminant of FBS that adheres to cells and leaches off during the conditioning of the medium. First, the material isolated from deficient FBS corresponding in size to component I (Fig. 4A) did not stimulate an in vitro primary response in cultures supplemented with deficient FBS (Table 1B). Second, there are cell lines grown under identical conditions to those under which ML-1, ML-2, and JLS-V5 cells were grown that do not secrete the factor (Table 2). TABLE 2. Leukemia virus-infected cell lines assayed for factor PFC/culture Origin of cell lines (1.2 X 107 input cells) Control (no factor) 310 Spleen lines ML-1 (BALB/c) 6840 ML-2 (BALB/c) 5230 JLS-V5 (BALB/c) 9760 Immunized spleen 3450 cells (BALB/c)* Plasmacytomas P1.17 (BALB/c) 410 P3.6.2 (BALB/c) 320 C1.18 (C3H) 740 Lymphomas S49 (BALB/c) 300 S117 (BALB/c) 240 Fibroblasts 3T3 (Swiss) 350 3T3-RLV (Swiss) 420 * Immunized spleen cell suspensions were prepared from mice injected intraperitoneally with sheep 3 days before culture. Cells were washed once in Eagle's medium without serum; 108 cells were incubated for 24 hr in 10 ml of the same medium. For other cell lines, 107 cells were incubated in 10 ml of Eagle's medium without serum. The medium was then removed from each culture and centrifuged successively at 2000 X g for 10 min and 100,000 X g for 90 min. The 50% ammonium sulfate precipitate was collected as described in Methods and dialyzed against Eagle's medium without serum; the insoluble precipitate was discarded after centrifugation. Each sample was sterilized by filtration through 0.45-,um Millipore filters and divided into three aliquots. Each aliquot was assayed for the factor in one spleen culture containing 1.2 X 107 cells in 1 ml of medium that contained deficient FBS and sheep. At day 4, each culture was assayed for PFC and the average PFC from the three aliquots is presented. -LJ U- 0D Regulation of In Vitro Immune Responses 597 w -J :) CL Io io3 B a- -a-_ a 100,Q a A /t A FIG. 5. (A) The kinetics of PFC production in spleen cell cultures containing 5% deficient FBS and supplemented with 0.5 (0), 5 (A), 25 (0), or 100 Mg (0) of component I, or with normal FBS (-). (B) Spleen cells in cultures containing 5% deficient FBS and sheep supplemented with 100 Mg of component I were collected, washed once, and suspended in fresh medium containing 5% deficient FBS, but no component I, at the times marked with arrows. The solid line represents the PFC response in control cultures supplemented with 100 Mg of component I; the broken line represents the PFC response in cultures where component I has been removed. The cell recoveries at the times indicated did not vary with the concentration of the factor present in the cultures. The factor found in supernatants from MI1, ML-2, or JLS-V5 cell cultures is not secreted by all cell lines infected with murine leukemia viruses. A number of plasmacytomas known to be infected with Gross-type leukemia viruses (9) and 3T3 cells infected with Rauscher leukemia virus do not secrete the factor. Uninfected spleen cells, however, do secrete the factor (Table 2). DISCUSSION At least three distinct cell types appear to be required for the production of an in vitro primary immune response to sheep : the lymphocytes derived from bone marrow or progenitor antibody-forming cells, lymphocytes, derived from thymus, and a third cell type referred to as adherent cells, because of their ability to adhere to glass or plastic surfaces (11-16). The contribution of accessory cell types to the differentiation of lymphocytes derived from bone marrow is difficult to determine in cultures of spleen cells, because only a small proportion of cells interact to initiate an immune response to any one antigen. Current studies of the interactions between these cell types are performed by partial removal of various cells by gradient centrifugation and adherence procedures, or by the irreversible inactivation of the expression of a cell type by specific antisera. The stimulation of a primary immune response by the factor secreted by M-1, MI2, and JLS-V5 cells in spleen cultures containing deficient FBS offers a new way of controlling the in vitro immune response. The deficient serum does not support the expression of one or more of the cell types required for the initiation of the immune response (Fig. 2). The concentration of factor in cultures containing deficient FBS controls both the length of time required for initiation of the primary immune response in vitro and the magnitude of the response. However, the rate of appearance of PFC seems to be independent of factor concentration (Fig. 5). Thus with saturating 5

5 598 Immunology: Watson and Thoman amounts of factor, the in vitro primary immune response in cultures containing deficient FBS appears identical to the immune response obtained with normal FBS. The manipulation of the initiation of the in vitro primary immune response by this factor makes it possible to study the expression of specific cell types in mixed spleen cell populations in a way that does not require the physical removal or irreversible inactivation of a specific cell type. The factor has also a useful practical application. The data discussed here report experiments with a specific batch of deficient FBS. All batches of FBS that do not support the initiation of an in vitro primary immune response in mouse spleen cultures, support responses in the presence of saturating amounts of factor. The factor can be used to overcome variability in the in vitro responses that arise from the use of different serum batches. It is unlikely that the factor is a product of infection of cells with murine leukemia viruses. A number of cell lines infected with Gross-type or Rauscher leukemia viruses do not secrete this factor (Table 2); normal spleen cells not infected with these viruses also secrete the factor. It is possible that Mbi1,.ML-2, and JLS-V5 cell lines have been derived from cell types the physiological function of 'which is to produce the factor and it is possible that leukemia virus infection has sustained factor production' in cultured cells. Leukemia viruses may prove useful biological tools for retaining the physiological function of cultured cells. There are several examples of hematopoietic and lymphoid cells infected with leukemia' viruses that maintain some aspects of their normal functions. Erythroleukemic - cells infected with Friend leukemia virus have been induced to differentiate along the erythroid pathway (17). Myelomas derived from BALB/c mice by oil-induction (18, 19) are bone marrow-derived lymphocytes infected with Gross-type leukemia viruses (9) that continue to synthesize antibody 'or maintain their differentiated state in the absence of antigenic and accessory cell stimulation. If Mb-1, ML-2, and JLS-V5 cells have been derived from cells whose physiological function is to secrete factor, then infection with leukemia viruses has resulted in a specific cell, which maintains a normal function. The factor secreted by macrophages (20) does not support the stimulation of a primary immune response in spleens cultured in deficient media (Hoffmann, M., personal communication). Many activities, possibly associated with different factors, have now been found in the culture supernatants of antigenically stimulated lymphocytes, certain of which induce DNA synthesis and cell division in lymphocyte cultures Proc. Nat. Acad. Sci. USA 69 (1972) (21, 22). The nature of such factors and their specificities is difficult to analyze because of the problem that multiple factors may be produced in any population of diverse lymphoid cell types. Other lymphoid tumors may continue to secrete various factors reflecting those produced by the corresponding cell type under physiological conditions. Such sources of factors are far more amenable to analysis than mixed lymphocyte populations. We thank Dr. R. I. Mishell for providing us with the batch number of the deficient FBS used and acknowledge the generous assistance provided by Dr. R. W. Dutton's laboratory in establishing the culture techniques for assay of the factor. This work was supported by a Ford Foundation grant no. 7505, a National Institute of Allergy and Infectious Diseases research grant no. A105875, and a National Institute of Allergy and Infectious Diseases training grant no. AI00430 to Dr. Melvin Cohn. 1. Mishell, R.' I. & Dutton, R. W. (1967) J. Exp. Med. 126, Nossal, G. J. 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