Immunology 1990 69 303-311

Growth of murine thymocytes in vitro in chemically defined medium G. W. WOOD, J. H. GREENWOOD & L. MAUSER Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas, U.S.A. Acceptedfor publication 12 September 1989

SUMMARY Immature T cells proliferate, diversify their repertoire of antigen specificity, are selected for MHCrestricted function, are selected for non-self reactivity and undergo maturation in the thymus. The mechanisms underlying thymic development are poorly understood. One reason for this is that murine thymocytes generally die when cultured in vitro under conditions which normally support lymphocyte growth. We describe conditions under which CD4-CD8- thymocytes proliferate at a high rate and acquire maturation-associated markers in vitro in the absence of exogenous mitogenic stimuli. CD4+CD8- cells also multiplied in the absence of added lymphokines while CD4-CD8+, but not CD4+CD8+, cells proliferated in the presence of exogenous IL-2. Proliferation of CD4-CD8- cells was associated with production of both IL-1 and IL-2. Proliferation of unfractionated, CD4-CD8- and CD4+CD8- thymocytes was dependent upon interaction of IL-2 with its receptor. CD4-CD8- cells acquired CD4 and/or CD8 markers during culture, indicating that, in addition to the proliferation, some maturation occurred. Proliferation occurred in complexes containing one or more central stromal cells. The results are discussed in relation to their possible relevance to thymocyte development. essential event in thymic development. Numerous investigations into possible cytokine-induced thymocyte proliferation have been performed, and IL-2 has emerged as a major candidate. The evidence for this is that: (a) a high proportion of both fetal (50%) and adult (30%) murine CD4-CD8- cells are IL-2R positive (Habu et al., 1985; Raulet, 1985; Ceredig, 1986); (b) anti-IL-2R antibody blocked thymocyte development in thymic organ culture (Jenkinson, Kingston & Owen, 1987) and in vivo (Tentori et al., 1988a); and (c) human Tl 1 +CD3-CD4-CD6-CD8- thymocytes and Ti 1 -CD3-CD4-CD6-CD8- prothymocytes are IL-2R positive (Toribio et al., 1986; DelaHera et al., 1986), can be stimulated with IL-2 to proliferate and differentiate into mature T cells in vitro (DelaHera et al., 1987) and produce and use IL-2 spontaneously during culture (DelaHera et al., 1987). Studies in vivo or using organ culture have provided considerable insight into thymic development. However, it is likely that development of culture systems using dissociated thymocytes is essential to increased understanding of the cellular and molecular events. We describe here a culture system wherein murine thymocytes undergo rapid proliferation in the absence of added polyclonal mitogens. Proliferation was dependent on IL-2 production. When cultures were initiated with CD4-CD8- cells, the cells acquired CD4 and/or CD8.

INTRODUCTION The T-cell portion of the murine immune system develops within the thymus by clonal expansion of small numbers of bone marrow-derived thymocyte precursors (Scollay & Shortman, 1984). Proliferation occurs principally in the subcapsular zone of the thymic cortex, but may occur elsewhere in the cortex. The CD4-CD8- subpopulation contains most, but possibly not all, of the proliferating (Mathieson & Fowlkes, 1984; Scollay, Bartlett & Shortman, 1984) cells and gives rise to the CD4+CD8+, CD4+CD8- and CD4-CD8+ subpopulations (Ceredig et al., 1983a; Ceredig, MacDonald & Jenkinson, 1983b; Fowlkes et al., 1985). Although there is some recent evidence that CD4-CD8- cells pass through a CD4+CD8+ stage during their differentiation into CD4+CD8- cells (Smith, 1987; Carbone, Marrack & Kappler, 1988; MacDonald, Hengartner & Pedrazzini, 1988; Fowlkes, Schwartz & Pardoll, 1988), little else is known about the pathways of maturation. As a result of the low number of precursors entering from the bone marrow, rapid proliferation of T-cell precursors is an Abbreviations: FCS, fetal bovine serum; IL-1, interleukin-l; IL-2, interleukin-2; IL-2R, interleukin-2 receptor; IL-4, interleukin-4; 2-ME, 2-mercaptoethanol; MLR, mixed lymphocyte reaction; NHS, normal human serum; NMS, normal mouse serum; PBS, Dulbecco's phosphate-buffered saline, pH 7-2. Correspondence: Dr G. W. Wood, Dept. of Pathology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS 66103, U.S.A.

MATERIALS AND METHODS Mice

Outbred Swiss/Webster mice were purchased from Harlan/ Sprague Dawley Inc. BALB/c and C3H strain mice were also

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G. W. Wood, J. H. Greenwood & L. Mauser

obtained from Harlan. The mice were maintained and bred in the animal care facilities of the University of Kansas Medical Center. Thymocytes Mice which were used as a source for thymus were between 2 and 6 weeks of age. They were killed by CO2 poisoning, the thoracic cavity was opened immediately and both lobes of the thymus were removed surgically and freed of contaminating blood by washing with PBS. Thymocyte cell suspensions were prepared either mechanically or enzymatically. Thymocytes were prepared mechanically by sieving minced thymus through stainless steel mesh into RPMI-1640 supplemented with penicillin and streptomycin, washing once with medium and performing viable counts. Mechanically prepared cells were used in a limited number of experiments to control for effects of collagenase digestion. All other experiments were performed with collagenase-dissociated thymus, because enzyme dissociation maximizes yield of both thymocytes and stromal cells and increases uniformity of cell preparation. To prepare thymocytes enzymatically, tissue was minced and suspended in type IV collagenase (0-5 mg/ml in PBS; Sigma Chemical Co.). The mixture was agitated at room temperature for 30 min, filtered through gauze to remove undigested fragments, sedimented at 200 g for 10 min, washed and counted.

Antibodies Monoclonal antibodies to CD4 [rat IgG2b (Wilde et al., 1983)], CD8 [rat IgM (Sarmiento, Glasebrook & Fitch, 1980)]; rat IgG2a (Ledbetter & Herzenberg, 1979), IL-2R [rat IgG (Mengle-Gaw et al., 1984)] and I-Ak [mouse IgG (Oi et al., 1978)] were obtained from the American Type Culture Collection. A clone producing antibody to IL-4 (O'hara & Paul, 1985) was generously provided by Dr W. E. Paul at the National Institutes of Health, Bethesda, MD. Cells were grown in RPMI-1640 supplemented with L-glutamine, antibiotics and 10% FCS. Antibodies were derived from tissue culture cell supernatants by 50% ammonium sulphate precipitation followed by 48 hr dialysis against PBS. The optimal dilutions of all antibodies were determined empirically by titration.

Lymphokines Highly purified recombinant human IL-2 from E. coli (Wang, Lu & Mark, 1984; Rosenberg et al., 1984) was generously provided by Cetus Corporation. IL-1 beta and IL-4 were generously provided by Immunex Corporation, Seattle, WA. Gamma-interferon was generously provided by the American Cancer Society. IL-1 beta was used at 1 nanogram/ml (0-2 ng/ well). IL-2 was used at 50 units/ml (12 5 units/well). IL-4 was used at 10 nanograms/ml (2-0 ng/well). Gamma-interferon was used at 100 units/ml (20 units/well).

Lymphokine quantification IL-1 was quantified by a modification of the standard thymocyte proliferation stimulation assay (Blyden & Handschumacher, 1977). The assay was modified in that IL-1 was quantified by its ability to directly augment thymocyte proliferation, rather than by augmentation of phytohaemagglutinininduced proliferation. Recombinant IL-l served as the positive control for the assays. Thymocytes were obtained from 4-week-

old BALB/c mice and cultured in RPMI- 1640 supplemented with antibiotics, 10% FCS, 1 x non-essential amino acids (Hazelton Biologicals), 1 mm Na pyruvate, 2 mm glutamine, and 5 x 10-5 M 2-mercaptoethanol. 1 5 x 106 cells were cultured in 100,l in 96-well microtitre plates. Test and control samples were added in 100 pl medium/well. Culture was continued for 72 hr, [3H]thymidine (1 pCi/well) was added for the last 8 hr, cells were harvested with a Brandel cell harvester and counted by liquid scintillation counting. IL-2 was quantified by its ability to stimulate the proliferation of the IL-2-dependent cell line, 53 (Rosenberg et al., 1984), generously provided by Dr Bernie Fox and Dr Steve Rosenberg at the National Institutes of Health. Recombinant IL-2 was used as positive control. Fifty-three cells were cultured in 96well microtitre plates at a concentration of 5 x 104/ml in 100 ml of medium; 100 pl of control and test samples were added to each well. The culture period was 24 hr. [3H]thymidine (1 pCi/ well) was added for the last 8 hr, cells were harvested with a Brandell cell harvester and counted by liquid scintillation counting.

Thymocyte subpopulation isolation CD4-CD8- cells were isolated by complement-dependent cytotoxicity with an anti-CD4, anti-CD8 cocktail. The specific cytotoxic depletion of cell subpopulations based on differential expression of CD4 and CD8 was performed as follows. Thymus was dissociated by enzymatic digestion. 1 x 107 thymocytes were mixed with a cocktail of anti-CD4 and anti-CD8 in 1 ml of RPMI-1640. The mixture was incubated for 15 min on ice, sedimented for 5 min at 200 g, resuspended in rabbit complement and incubated for 30 min at 370 to kill antigen-positive cells. At the end of 30 min the mixture was layered onto Histopaque (specific density 1 1; Sigma Chemical Co.) and sedimented at 2000 r.p.m. for 10 min. Cells were removed from the interface and washed with RPMI-1640. To ensure removal of antigen-positive cells, the entire treatment was repeated. In some experiments, to determine the effect on proliferation of removal of cells expressing low levels of CD4 and CD8, the cytotoxicity-depleted CD4-CD8- population was reincubated with IgG anti-CD4 and IgG anti-CD8, and panning was performed on plates coated with anti-rat IgG as described previously (Wysocki & Sato, 1978). This step removed cells which were coated with antibody, but which had not been killed. CD4-CD8- cell populations isolated by either method contained less than 0 5% contamination by CD4+ or CD8+ cells as assessed both by flow cytometry and immunocytochemistry with the appropriate antibodies. Never the less, as has been the case in all studies of CD4-CD8- thymocytes, double negativity is operationally defined. The CD4-CD8- cells may still have expressed CD4 and/or CD8, but it was below the level of sensitivity of the assays employed. CD4+CD8+ cells were isolated by sequential specific panning. Cells were coated with rat IgG anti-CD4 and rat IgM antiCD8 at 40, then adhered to polyclonal anti-rat IgG coated plates at 4°. Non-adherent cells were washed off, and the attached CD4+ cells were collected and added to polyclonal anti-rat IgMcoated plates to positively select for CD8+ cells, which, in this case, should only have been CD4+CD8+. The adherent CD4+CD8+ cells then were collected. CD4+CD8- cells were isolated by a combination of specific cytotoxicity and panning. First, CD8+ cells were removed by

Murine thymocyte proliferation in vitro complement-dependent cytotoxicity with rat IgM anti-CD8. Second, CD4+ cells were obtained by coating remaining cells with rat IgG anti-CD4, panning on polyclonal anti-rat IgGcoated plates and isolating adherent cells. The latter step separated the single positive cells from the double negative cells. CD4-CD8+ cells also were isolated by a combination of cytotoxicity and panning. First, CD4+ cells were depleted by complement-dependent cytotoxicity with rat IgG anti-CD4. Second, CD8+ cells were obtained by coating remaining cells with rat IgM anti-CD8, adding cells to polyclonal anti-rat IgMcoated plates, and isolating adherent cells. All panning was performed in the presence of 4-0 mg/ml human IgG as an Fc receptor blocker. Because the subpopulations were isolated using the same antibodies that would be used to characterize cell purity, determining the level of contamination with other cell types by serological analysis of the derived cell populations was never achieved. The essential step in isolation of all but the CD4-CD8- cells was panning. Great care was always taken that the adherent cells were resistant to excessive washing. In fact, in all experiments, the adherent cells had to be detached using teflon scrapers. Despite the absence of independent criteria for assessing the purity of the thymocyte subpopulations, the fact that each functioned in a distinctive fashion in culture argues that they were successfully isolated (see the Results and Discussion). Cellular phenotyping Indirect immunofluorescence was employed in these assays. In brief, 5 x 106 cells in 100 y1 were mixed with 100 p1 of monoclonal antibody on ice for 30 min. Cells were washed twice in ice-cold PBS, then 100 yl of fluoresceinated anti-rat IgG was added and the cells reincubated for 30 min on ice. Cells were washed and fixed with paraformaldehyde. Positive cells were identified and fluorescent intensity determined by flow cytometry using a Coulter EPICS. All reagents were suspended in 0-1% bovine serum albumin (BSA) and 0 I1% sodium azide in Hanks' balanced salt solution containing lI0 mg/ml human IgG to block Fc receptor binding. Both anti-CD4 and anti-CD8 were purified rat IgG monoclonal antibodies obtained from BectonDickinson, Mountain View, CA. The fluoresceinated affinitypurified anti-rat IgG secondary reagent also was obtained from Becton-Dickinson.

Thymocyte culture The medium used in most of the experiments was KC2000 (Hazelton Research Products Inc., Lenexa, KS). KC2000 is made up of a base of salts, vitamins and amino acids and a supplement containing L-glutamine, antibiotics, 2-ME, fetuin, transferrin and albumin. The final concentration of the protein components was less than 250 yg/ml. In selected experiments, the KC2000 was replaced with Nutridoma-HU, a serum-free medium (Boehringer-Manheim, Indianapolis, IN) which contains bovine insulin, human transferrin, human albumin, vitamins, amino acids and other small organic molecules. Nutridoma has a final protein concentration of less than 40 yg/ml. It was prepared in RPMI-1640 containing L-glutamine, antibiotics and 5 JM 2-ME. Other media used in these experiments were RPMI-1640 supplemented with L-glutamine, 5 yM 2-ME, antibiotics and 10% FCS, 2 5% normal human serum or 2-5% normal mouse serum. Dissociated thymocytes were washed once with KC2000 and

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1 X 106 cells in 100 yl of KC2000 were placed in each well of 96well microtest plates. Cells were cultured at 370 in a humidified atmosphere of 5% Co2 and 95% air. [3H]thymidine (1 yCi/well) was added 8 hr prior to termination of culture. Cells were harvested with a Brandel Cell Harvester and counted by liquid scintillation. Autoradiography Acid-cleaned slides were subbed (dipped in gelatin) and [3H]thymidine (ICN, 6-7 Ci/mMole) labelled cells were cytospun onto the coated slides. Slides were dipped in emulsion and exposed for 48 hr in black boxes at 4°. Slides then were developed using Kodak D-19 developer and fixed with Kodak acid fixer. Slides were counter-stained by the Wright's method and assessed visually for cells having silver grains over their nucleus.

RESULTS Demonstration of thymocyte proliferation in chemically defined medium T lymphocytes from adult mice proliferate maximally in response to antigenic challenge in medium containing serum. Extensive studies in this and other laboratories have established optimal conditions for such proliferative responses. One-way allogeneic MLRs performed during this study were maximal when cells were cultured in RPMI-1640 supplemented with L-glutamine, antibiotics, 2 5% normal human serum or 2-5% normal mouse serum and 5 yM 2-ME (Table 1). Thymocytes do not proliferate in serum-containing media unless polyclonal mitogens are added (Table 1). Recently, we performed experiments to determine whether or not chemically defined media would support antigen-specific responses. When MLRs were Table 1. Comparison of chemically defined medium with serumcontaining medium in supporting lymphocyte proliferation

Spleen cells C3H responders alone C3H x BALB/c MLR Unfractionated thymocytes Day 1 Day2 Day 3 Day 4 Day 5 Day 6 CD4-CD8- thymocytes Day 5

KC2000*

RPMI-1640 2.5% NHS

(c.p.m.)

(c.p.m.)

21,550+1187 139,036± 17,268

9049+1296 71,435+4025

1365+120 871+120 3483+985 6443+1189 11,810+1549 19,554+919

1992+225 539+79 151±33 76+19 417+103 89+41

164,431±+10,580

16,989+524

* Cells were grown in 96-well tissue culture plates. 1 x 106 cells were cultured/well in 100 p1. An 8-hr pulse of [3HJthymidine was given at the end of the culture period, cells were harvested and counted in a scintillation counter. Thymocytes all were obtained from 2-6-week-old Swiss-strain mice. MLRs were performed with 5 x 105 irradiated BALB/ c spleen cells as stimulators mixed with 5 x 105 C3H spleen cells as responders. Cells were cultured for 4 days and harvested.

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G. W. Wood, J. H. Greenwood & L. Mauser zi9,) _ 8 6-

Table 2. Proliferation by Swiss-strain thymocytes in different media

Thymocytes

Medium

Proliferation (c.p.m.)*

CD4- CD8CD4+ CD8+

0x 114

KC2000 KC2000 Nutridoma Nutridoma RPMI, 2 5% NMS RPMI, 2-5% NMS

9310 +942 162,607 + 10,992 12,270+ 1227

111,796+9593

O-O-/I 0-0

D-Ha

\

CD4+ CD8

I:2

Swiss Unfract. Swiss CD4-CD8Swiss Unfract. Swiss CD4-CD8 Swiss Unfract. Swiss CD4-CD8-

Thymocyte populations:

Unfroctionated

110-

A

CD4- CD8+ A-A

a) a-

8-

e c :3

6_

4568 + 1148

26,020±860

2

-

0

Cells were grown in 96-well tissue culture plates. 1 x 106 cells were cultured/well in 100 pl. An 8-hr pulse of [3H]-thymidine was given after 112 hr of culture, cells were harvested and counted in a scintillation counter. *

2

0

3

4

5

8

Days in culture

Figure 1. Proliferation by Swiss-strain thymocytes in KC2000. Kinetics of proliferation by CD4-CD8- and unfractionated cells.

performed in KC2000, a high level of proliferation in response to MHC differences was sustained (Table 1). Interestingly, unstimulated responder cells proliferated to a higher degree in

KC2000 than in serum-containing medium, while antigenspecific MLRs were highest in the serum-containing medium. Thus, stimulation indices were higher in medium containing serum (Table 1). Because of the high background proliferation, we hypothesized that chemically defined media might support thymocyte proliferation. When murine thymocytes from Swiss/Webster mice were cultured in KC2000, a high degree of proliferation was observed (Table 1). The same cells failed to proliferate at comparable levels in medium supplemented with mouse, human or fetal bovine serum (Table 1). A second chemically defined medium, containing a lower concentration of protein (KC2000

Growth of murine thymocytes in vitro in chemically defined medium.

Immature T cells proliferate, diversify their repertoire of antigen specificity, are selected for MHC-restricted function, are selected for non-self r...
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