- Email: [email protected]
− double-knockout mice
Immunology Letters 67 (1999) 243 – 249
Generation and function of bone marrow-derived dendritic cells from CD4/CD8 − / − double-knockout mice Massimiliano M. Corsi a, Johan K. Sandberg b, Ken Wasserman b, Hubert H. Maes b, Rolf Kiessling b,c,* b
a Institute of General Pathology, Faculty of Medicine, Uni6ersity of Milan, Milan, Italy Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden c Department of Oncology, Radiumhemmet, Karolinska Hospital, Stockholm, Sweden
Received 11 January 1999; accepted 1 February 1999
Abstract We present a novel, simple and straightforward method to obtain mouse bone marrow-derived dendritic cells (DC) from C57Bl/6 CD4/CD8 − / − double knock-out mice. This new method, involving culture of bone marrow cells in medium supplemented with Interleukin 4 and Granulocyte-Macrophage Colony-Stimulating Factor, does not involve negative immunodepletion of CD4 + and CD8 + populations, or extensive prior manipulations of the starting population. The resulting, loosely adherent cell population, exhibited the morphological characteristics and typical surface markers of DCs, and were endowed with the functional activities characteristic of bone marrow-derived DCs of wild-type mice. Interestingly, LCMV GP33-41 peptideloaded CD4/CD8 − / − DCs were efficiently lysed by peptide-specific activated CTLs in vitro. Furthermore, these peptide-loaded CD4/CD8 − / − DCs induced a peptide-specific CTL response upon immunization of wild-type C57Bl/6 mice. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; CD4/CD8 double knockout mice; mrGM-CSF; mouse recombinant IL-4; CTL-mediated DC lysis
1. Introduction Since they were described [1], dendritic cells (DC) have been under intense investigation because they are antigen-presenting cells able to both induce potent primary immune responses [2 – 6], and to facilitate regression and elimination of established tumor burdens in mice [7]. Intriguingly, striking differences in DC potency were observed depending upon the culture conditions employed. Only tumor peptide-pulsed DC Abbre6iations: CTL, cytolytic T lymphocytes; DC, dendritic cells; GM-CSF, granulocyte-macrophage colony stimulating factor; IL-x, interleukin x; LCMV, lymphocytic choriomeningitis virus; RMA-S, antigen processing defective mutant from the murine RMA lymphoma. * Corresponding author. Tel.: +46-8-7286688; fax +46-8-309195. E-mail address: [email protected] (R. Kiessling)
stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), but not DC cultured in either GM-CSF alone or GM-CSF and tumor necrosis factor alpha (TNF-a) were capable of inducing tumor regression [7]. Established protocols for the growth of DC from mouse bone marrow [8] typically involve extensive depletion steps in order to culture purified populations, and thereby avoid the undesirable proliferation of other cell types. We therefore aimed at developing a simpler method of murine DC culture, as we previously did in a human system [9]. We sought to eliminate depletion steps and take advantage of the availability of CD4/CD8 − / − double knockout mice, which have been obtained by extensive screening of the offspring of heterozygous CD4 + / − CD8 + / − mice [10]. Although these mice are deficient in both helper and class-I-restricted cytotoxic activity,
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they can mount allograft rejection and CD1-restricted responses involving antigen presentation by DC cells. In our work, we utilized bone marrow from CD4/ CD8 − / − mice to generate DCs devoid of contaminating CD4 and CD8 positive cells. Furthermore, we tested the functionality of these CD4/CD8 − / − DC as target cells for peptide specific CTL, and inducers of CTL in wild-type mice. Our results have implications for the use of DCs as antigen presenting cells in future vaccine development.
2. Materials and methods
2.1. Animals and cell lines The C57BL/6 double (CD4/CD8) knock-out mice were bred and maintained in the pathogen-free animal facility of the Tumor Biology and Microbiology Center, Karolinska Institute (MTC, KI).The wild-type C57Bl/6 mice were purchased from and maintained in the normal animal facility of the MTC, KI. RMA-S is an antigen processing-(TAP deficient) and presentation-deficient mutant of a Rauscher leukemia virus-induced T-cell lymphoma RBL-5 of C57Bl/6 origin [11]. Expression of MHC-I molecules at its surface can be increased when cultured in cold conditions and stabilized by addition of endogenous peptide [12].
2.2. Media and cytokines Mouse dendritic cell (DC) culture medium was composed of RPMI 1640 supplemented with 10% fetal calf serum (FCS), 5× 10 − 5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ ml streptomycin. Murine CTL culture medium was composed of MEM-a supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 5× 10 − 5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. All media and supplements were purchased from Gibco BRL (Life Technologies, Sweden). Mouse recombinant IL-4 (mrIL-4) was kindly donated by Schering-Plough Research Institute (Kennilworth, NJ, USA). Mouse recombinant GM-CSF (mrGM-CSF) was purchased from ImmunoKontact (Frankfurt am Main, Germany). Human recombinant IL-2 (hrIL-2) (Dupont) and mouse recombinant IL-12 (mrIL-12) (Hoffman-La Roche) were kindly provided by their respective manufacturers.
2.3. De6elopment of dendritic cells from bone marrow All mice were sacrificed by lethal exposure to ether and then subjected to cervical vertebrae dislocation. Femurs, tibia, ulnae and radii were obtained from
wild type or CD4/CD8 − / − mice. Muscle tissue was removed and bones were placed in a 15 ml tube containing 70% alcohol for 1 min. The bones were then serially transferred two times into 15 ml sterile PBS before the epiphyses were cut using scissors. Marrow tissue was expelled from both the dyaphysis and the epiphyses by fluid pressure using a needle and supplemented DC culture medium. The marrow of approximately one femur diluted in 2 ml medium was placed directly into each well of a 24 well cell culture plate (Costar). Then 1000 IU/ml of both mrGM-CSF and mrIL-4 were added and cells were incubated at 37°C, 7.2% CO2. On days 1 and 3 post culture initiation, the contents of each well were divided in half and the volume of each new well was brought to 2 ml with supplemented DC medium and mrGM-CSF+mrIL-4 (1000 IU/ml final each). On day 6–7 the same procedure was repeated for the final time, but using only 500 IU/ml mrGM-CSF and 1000 IU/ml mrIL-4. Overall cell morphology was examined every day by phase contrast microscopy.
2.4. hrIL-2 /mrIL-12 Cultured bone marrow cells For comparative purposes, CD4/CD8 − / − bone marrow cultures stimulated by an hrIL-2+ mrIL-12 combination were also established. Extraction of bone marrow was performed as described above. After overnight incubation (37°C, 7.1% CO2) of the cells in supplemented DC medium, plastic non-adherent bone marrow cells (2× 106/ml) were removed by pipetting and incubated in supplemented DC medium containing 500 IU/ml hrIL-2 (a generous gift from Dupont) and 0.5 ng/ml mrIL-12 (a generous gift from Hoffman-La Roche). On day 3 of culture, the medium was removed by centrifugation, and replaced by 5 ml supplemented DC medium containing fresh hrIL-2 and mrIL-12 as above.
2.5. Flow cytometry Analysis was performed on day 9 of cell culture. Cells (5× 105) were washed in PBS containing 1% FCS and incubated for 20 min on ice with the primary antibody as indicated. The following primary mAbs (10 mg/ml) were used: anti-B7.1 (IG10) and anti-B7.2 (GLI) (purchased from Pharmagen and kindly provided by Dr Hans Gustaf-Ljungren, MTC, Karolinska Institute), anti-ICAM 1 and anti-LFA-1 (donated by Dr Manuel Pattaroyo MTC, Karolinska Institute), anti-IA (donated by Dr Staffan Pauli, Department lmmunology, Stockholm University). An irrelevant primary antibody, anti IgG1 (Dako) was used as a control. Washed cells (2×) were then incubated on ice for 20 min with either a secondary fluorescein-conjugated anti-mouse IgG F(ab)2, at a
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1:50 dilution, or streptavidin-FITC, as directed by the manufacturer (DAKOPATTS, AB) and washed again. In all cases, washing solution was composed of PBS containing 1% FCS. For each sample, 10 000 events were acquired using a FACScan (Becton Dickinson) and setting forward and side scatter gating to exclude cell debris.
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was calculated according to the formula: % specific release=((experimental release− spontaneous release)/ (maximum release− spontaneous release))× 100.
3. Results
3.1. De6elopment of dendritic cells 2.6. Generation and culture of 6irus-specific CTL To generate a LCMV (lymphocytic choriomeningitis virus) GP 33-41-specific CTL line, the synthetic peptide corresponding to the LCMV GP 33-41 epitope was synthesized by a solid phase method using F-moc solid phase chemistry, and analyzed by reverse phase HPLC and electrospray mass spectrometry. Wild-type C57Bl/6 mice were inoculated i.p., with 102 pfu LCMV. Two weeks after immunization, 25× 106 immune spleen cells were cocultured with 25× 106 irradiated C57Bl/6 spleen cells in the presence of 0.1 mM LCMV GP 33-41 peptide in 12 ml supplemented CTL medium (37°C, 5% CO2). Every 10 days, the CTL line was restimulated with 10 × 106 irradiated and peptide-coated splenocytes in supplemented CTL medium containing 20 IU/ml IL-2. These cells were then used as effectors in cytolytic assays against CD4/ CD8 − / − DC or RMA-S cells loaded or not with the GP 33-41 peptide.
2.7. Immunization using peptide-coated DC DC from C57Bl/6 or CD4/CD8 − / − mice were incubated at 37°C with 30 mM LCMV GP 33-41 peptide for 2 h in serum-free DMEM, washed twice, and injected subcutaneously [13]. Ten to thirteen days after immunization, 25×106 immune spleen cells were isolated and cocultured with 25×106 irradiated autologous spleen cells in the presence of 0.1 mM peptide in 12 ml supplemented CTL medium at 37°C, 5% CO2. Five days later, these cells were used as effectors in CTL assays against RMA-S cells loaded or not with the GP 33-41 peptide.
2.8. CTL assay CTL activity was measured using a standard 51Crrelease assay. Briefly, RMA-S cells or DC from C57Bl/6 CD4/CD8 − / − mice were used as targets. Target cells were labeled with 100 ml of a 1 mCi/ml 51 Cr solution for 1 h at 37°C. Cells were then coated with titrated amounts of peptide (10 − 3 – 10 − 8 mM) for 2 h. Cells were further washed three times before being incubated (3×103 target cells/well) with various amounts of effector cells (E:T ratios of 45:1, 15:1, 5:1, 2:1) for 4 h at 37°C, 5% CO2. After incubation, released radioactivity was measured and specific lysis
Murine recombinant GM-CSF and IL-4 were added at the inception of in vitro cultures as described in the methods. Following overnight incubation at 37°C, CD4/CD8 − / − bone marrow cells formed a mosaic of small round cells which surrounded occasional clumps of bone marrow tissue beneath small mats of agglutinated red blood cells (Fig. 1A). By day 2, small islets of slightly larger cells among the occasional adherent cells could be observed (Fig. 1B). By day 7, prominent islets of cells were readily observed (Fig. 1C) loosely attached to fibroblast-like cells which adhered to tissue-culture treated wells (Fig. 1D).
3.2. Flow cytometric analysis of cells deri6ed from CD4 /CD8 − / − bone marrow cultures Loosely attached cells from cultures depicted in Fig. 1C were readily obtained by gentle resuspension via pipetting and subjected to immunofluorescent analysis, as presented in the materials and methods. For comparison, bone marrow cells of the same origin, but cultured in the presence of hrIL-2 and mrIL-12 (LAK cells) [14], were treated in parallel with the same combinations of primary and secondary reagents. Relative to the negative control cells, CD4/CD8 − / − bone marrow DC (BMDC) expressed clearly elevated levels of cell-surface markers often associated with differentiated/activated DC, such as IA, CD80, CD86, ICAM-1 and LFA-1 (Fig. 2). Note, the two distinct populations of IA + and CD86 + DC cells. In all cases, but the irrelevant primary antibody used as a control, the expression of these five markers characteristic for differentiated/activated DC cells were higher in DC than in LAK cells.
3.3. CD4 /CD8 − / − DCs are efficient antigen presenting cells The functional characteristics of the CD4/CD8 − / − DC cells were tested by two distinct methods. First, the CD4/CD8 − / − DCs were used as target cells for peptide-specific CTLs: CD4/CD8 − / − DCs and RMAS cells were loaded with the H-2Db restricted LCMV GP33-41 peptide, and tested as target cells for GP3341 specific CTLs in a 51Cr-release cytotoxicity assay (Fig. 3). The results indicated that peptide-pulsed
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CD4/CD8 − / − DCs were lysed just as efficiently as peptide-pulsed RMA-S cells, demonstrating that CD4/ CD8 − / − DCs are efficient antigen presenting cells in vitro. Second, the antigen presenting capacity of CD4/ CD8 − / − DCs in vivo [15] was examined. DCs generated from CD4/CD8 − / − and wild type C57Bl/6 bone marrow cultures were incubated in vitro with the LCMV GP33-41 peptide before being injected s.c. in C57Bl/6 mice, as described in the materials and methods section. Ten to thirteen days after immunization, mice were sacrificed and the isolated splenocytes were restimulated 5 days in vitro with autologous splenocytes and the relevant peptide. Resulting CTL activity was tested in a 51Cr-release cytotoxicity assay against RMA-S cells loaded with the LCMV GP33-41 peptide (Fig. 4). The CTLs from mice immunized with either CD4/CD8 − / − or C57Bl/6 control DCs were both peptide specific, and exhibited a somewhat similar cytolytic activity although that of wild type DC was higher than that of knock-out DC. Together, these results indicate that DCs from CD4/ CD8 − / − bone marrow cultures are functionally competent professional antigen presenting cells: they are both able to induce peptide-specific CTL responses and be recognized by peptide specific CTLs. 4. Discussion In view of the important role DC have been found to have in generating antigenic responses [2,3,6], it is of
particular interest to generate both large quantities and purified populations of DC for both research and therapeutic uses. Previous workers have already developed methods to generate large numbers of DC, but the isolation of purified populations or sub-populations of DC are tedious and not always sufficient to guarantee exclusion of contaminant cells that may influence the results [3,8,16]. Here, we first wanted to determine if the same culture supplements used previously with human DC [9,17] would also be adequate for murine DC development. We found that bone-marrow derived CD4/CD8 − / − DC cells cultured as described here exhibit a number of markers that are found on activated/differentiated DC cells (Fig. 2) [6]. The presence of five of them together (IA, CD80, CD86, ICAM-1 and LFA-1), although not individually specific to DC, together with the characteristic morphological appearance of these cells (Fig. 1), permit us to state that bone marrow cells cultured in the regimen of rmIL-4 and rmGM-CSF described here lead to the development of DC cells. Second, we aimed at generating DC cultures totally free of interfering CD3, CD4 and CD8 lymphocytes. We thus developed a methodology to develop DC from CD4/CD8 − / − mice. CD4/CD8 − / − double-knock-out mice were chosen because the overall numbers of CD3 + T cells among bone marrow cells from CD4/ CD8 − / − double knock-out mice are extremely low [10]. Very low initial numbers of contaminating cells coupled with conditions favoring the outgrowth of DCs allowed for the outgrowth of virtually pure DC cultures
Fig. 1. Phase contrast microscopic analysis of CD4/CD8 − / − C57B1/6 mouse bone marrow cultures stimulated with rmGM-CSF and rmIL-4. A, appearance on day1; B, small islets of cells on day 2; C, large islets of loosely adherent cells on day 7; D, underlying stromal cells cleared of loosely adherent DCs on day 7. Bar =25 mm.
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Fig. 2. Flow cytometric analysis of the 9 day-old loosely adherent subpopulation derived from bone marrow cultures stimulated with rmGM-CSF and rmIL-4. Bold lines, bone marrow-derived DC; normal line, bone marrow-derived LAK cells, dotted lines, bone marrow-derived DC reacted with an irrelevant primary antibody (IgG1) as control. Expression of: A, IA; B, CD 80; C, CD 86; D, ICAM-1; E, LFA-1; F, negative control, irrelevant primary antibody (IgG1).
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Fig. 3. Cytolytic activity of anti-LCMV GP 33-41 CTLs against LCMV GP 33-41-loaded CD4/CD8 − / − (circles) and LCMV GP 33-41-loaded RMA-S cells (inverted triangles), both at an E:T ratio of 20:1 (black) and 4:1 (clear). Loading peptide concentrations are displayed on the X-axis.
without extensive depletion steps or other manipulations. The recognition of CD4/CD8 − / − DCs obtained by our method of culture by CTLs and their ability to mount an adequate CTL response were demonstrated (Figs. 3 and 4), indicating that these DCs are functional. Results of these assays also permit us to advance other hypotheses as to the requirements of DC develop-ment. The DCs generated by our method induced a peptide specific CTL response upon immunization of wild-type C57Bl/6 mice (Fig. 4). This indicates that CD8 + and CD4 + cells are not necessary to support development of functional DCs in the bone marrow cultures and that the CD8 and CD4 gene products are not necessary for proper DC development. Remarkably, the CD4/CD8 − / − DCs incubated with the specific MHC class I restricted peptide were sensitive targets for activated CTLs (Fig. 3). These data suggest that CD4/CD8 − / − DCs are fully competent antigen presenting cells and are able to induce vigorous CTL responses. Kronin et al. [18] suggested that the CD8 + subpopulation of DCs have immuno-regulatory properties. Indeed, his data indicated that CD8 + DCs inhibited proliferation of CD8 + CTLs. We therefore expected the CTL responses induced by CD4/CD8 − / − DCs to be stronger than those induced by mixed populations of wild-type DC. Our results indicate rather that wild type DCs are slightly better at inducing CTL re-
sponses. Our data nevertheless do not exclude an immunoregulatory role for the CD8 + DC-subpopulation. Future studies on the immunobiology of CD4/CD8 − / − DCs are required to shed light on this issue. DC cells isolated from CD4/CD8 − / − cultures are thus of particular interest to study the immune responses independent of helper T cells, the importance of DC cells as APC in general, the mechanisms of CD1-restricted responses and the roles played by CD4 + and CD8 + subpopulations [19,20]. In conclusion, we have developed a simple way to generate pure DC cultures from CD4/CD8 − / − double knockout mouse bone marrow progenitors by supplementing cultures with rmGM-CSF and rmIL-4. The resulting cultures were composed of loosely adherent cells easily separated from the underlying strongly adherent stromal cells by gentle pipetting. These bone marrow-derived loosely adherent cells were found to have the overall morphology of DCs and exhibited cell surface markers characteristic of activated/differentiated DC. Functional assays indicated that these cells, when loaded with a MHC class I restricted peptide epitope, were able to elicit a cellular immune reaction in vivo, and to serve as antigen presenting cells in vitro. Our data and technique should prove helpful in the understanding of DC biology.
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.
Fig. 4. Ability of DC to elicit a peptide-specific CTL response. DC cells derived from wild type (circles) or CD4/CD8 − / − (squares) C57Bl/6 bone marrow were loaded with the LCMV GP 33-41 peptide before being injected in wild type C57Bl/6 mice. Ten to 13 days later, spleens were removed and splenocytes restimulated with peptide in the presence of autologous irradiated splenocytes. RMA-S cells, loaded (black) or not (clear) with the LCMV GP 33-41 peptide, were then used as target cells in two separate experiments (right and left panels). The E:T ratios are displayed on the X-axis.
Acknowledgements This work was supported by grants from The Swedish Cancer Society, The Cancer Society of Stockholm, and the King Gustaf V jubilee fund. We thank L. Szekely, MD, for the microscope pictures and A. Fulgenzi, B.Sc., for his help in type-writing the manuscript.
References [1] R.M. Steinman, Z.A. Cohn, J. Exp. Med. 137 (1973) 1142 – 1162. [2] K. Inaba, R.M. Steinman, Science 229 (1985) 475–479. [3] S. Grabbe, S. Beissert, T. Schwartz, R.D. Granstein, Immun. Today 16 (1995) 116–120. [4] B. Mukherji, N.G. Chakraborty, S. Yamasaki, et al., Proc. NatI. Acad. Sci. USA 92 (1995) 8078–8082. [5] F.J. Hsu, C. Benike, F. Fagnoni, et al., Nature Med. 2 (1996) 52 – 58. [6] D.N.J. Hart, Blood 90 (1997) 3245–3287. [7] J.I. Mayordomo, T. Zorina, W.J. Storkus, et al., Nature Med. 1 (1995) 1297 – 1302.
[8] K. Inaba, M. Inaba, N. Romani, et al., J. Exp. Med. 176 (1992) 1693 – 1702. [9] K. Wasserman, M.M. Corsi, L. Szekely, K. Kono, H. Maes, R. Kiessling, Scand. J. Immunol. 45 (1997) 678 – 682. [10] M.W. Schilham, W.P. Fun – Leung, A. Rahemtulla, et al., Eur. J. Immunol. 23 (1993) 1299 – 1304. [11] L. Franksson, M. Petersson, R. Kiessling, K. Ka¨rre, Eur. J. Immunol. 23 (1993) 2606 – 2613. [12] H.G. Ljunggren, N.J. Stam, C. O8 hlen, et al., Nature 346 (1990) 476 – 480. [13] A. Porgador, D. Snyder, E. Gilboa, J. Immunol. 156 (1996) 2918 – 2926. [14] A. G. Lamont, L. Adorini, Immunol. Today 17 (1996) 214–217. [15] P. Brossart, M.J. Bevan, Blood 90 (1997) 1594 – 1599. [16] L. Lu, W.A. Rudert, S. Qian, et al., J. Exp. Med. 182 (1995) 379 – 387. [17] S.M. Kiertscher, M.D. Roth, J. Leukoc. Biol. 59 (1996) 208– 218. [18] V. Kronin, D. Vremec, K. Winkel, et al., Int. Immunol. 9 (1997) 1061 – 1064. [19] D.I. Gabrilovich, S. Nadaf, J. Corak, J.A. Berzofsky, D.P. Carbone, Cell. Immunol. 170 (1996) 111 – 119. [20] V. Flamand, T. Sornasse, K. Thielemans, et al., Eur. J. Immunol. 24 (1994) 605 – 610.
Generation and function of bone marrow-derived dendritic cells from CD4/CD8 − / − double-knockout mice Massimiliano M. Corsi a, Johan K. Sandberg b, Ken Wasserman b, Hubert H. Maes b, Rolf Kiessling b,c,* b
a Institute of General Pathology, Faculty of Medicine, Uni6ersity of Milan, Milan, Italy Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden c Department of Oncology, Radiumhemmet, Karolinska Hospital, Stockholm, Sweden
Received 11 January 1999; accepted 1 February 1999
Abstract We present a novel, simple and straightforward method to obtain mouse bone marrow-derived dendritic cells (DC) from C57Bl/6 CD4/CD8 − / − double knock-out mice. This new method, involving culture of bone marrow cells in medium supplemented with Interleukin 4 and Granulocyte-Macrophage Colony-Stimulating Factor, does not involve negative immunodepletion of CD4 + and CD8 + populations, or extensive prior manipulations of the starting population. The resulting, loosely adherent cell population, exhibited the morphological characteristics and typical surface markers of DCs, and were endowed with the functional activities characteristic of bone marrow-derived DCs of wild-type mice. Interestingly, LCMV GP33-41 peptideloaded CD4/CD8 − / − DCs were efficiently lysed by peptide-specific activated CTLs in vitro. Furthermore, these peptide-loaded CD4/CD8 − / − DCs induced a peptide-specific CTL response upon immunization of wild-type C57Bl/6 mice. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; CD4/CD8 double knockout mice; mrGM-CSF; mouse recombinant IL-4; CTL-mediated DC lysis
1. Introduction Since they were described [1], dendritic cells (DC) have been under intense investigation because they are antigen-presenting cells able to both induce potent primary immune responses [2 – 6], and to facilitate regression and elimination of established tumor burdens in mice [7]. Intriguingly, striking differences in DC potency were observed depending upon the culture conditions employed. Only tumor peptide-pulsed DC Abbre6iations: CTL, cytolytic T lymphocytes; DC, dendritic cells; GM-CSF, granulocyte-macrophage colony stimulating factor; IL-x, interleukin x; LCMV, lymphocytic choriomeningitis virus; RMA-S, antigen processing defective mutant from the murine RMA lymphoma. * Corresponding author. Tel.: +46-8-7286688; fax +46-8-309195. E-mail address: [email protected] (R. Kiessling)
stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), but not DC cultured in either GM-CSF alone or GM-CSF and tumor necrosis factor alpha (TNF-a) were capable of inducing tumor regression [7]. Established protocols for the growth of DC from mouse bone marrow [8] typically involve extensive depletion steps in order to culture purified populations, and thereby avoid the undesirable proliferation of other cell types. We therefore aimed at developing a simpler method of murine DC culture, as we previously did in a human system [9]. We sought to eliminate depletion steps and take advantage of the availability of CD4/CD8 − / − double knockout mice, which have been obtained by extensive screening of the offspring of heterozygous CD4 + / − CD8 + / − mice [10]. Although these mice are deficient in both helper and class-I-restricted cytotoxic activity,
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they can mount allograft rejection and CD1-restricted responses involving antigen presentation by DC cells. In our work, we utilized bone marrow from CD4/ CD8 − / − mice to generate DCs devoid of contaminating CD4 and CD8 positive cells. Furthermore, we tested the functionality of these CD4/CD8 − / − DC as target cells for peptide specific CTL, and inducers of CTL in wild-type mice. Our results have implications for the use of DCs as antigen presenting cells in future vaccine development.
2. Materials and methods
2.1. Animals and cell lines The C57BL/6 double (CD4/CD8) knock-out mice were bred and maintained in the pathogen-free animal facility of the Tumor Biology and Microbiology Center, Karolinska Institute (MTC, KI).The wild-type C57Bl/6 mice were purchased from and maintained in the normal animal facility of the MTC, KI. RMA-S is an antigen processing-(TAP deficient) and presentation-deficient mutant of a Rauscher leukemia virus-induced T-cell lymphoma RBL-5 of C57Bl/6 origin [11]. Expression of MHC-I molecules at its surface can be increased when cultured in cold conditions and stabilized by addition of endogenous peptide [12].
2.2. Media and cytokines Mouse dendritic cell (DC) culture medium was composed of RPMI 1640 supplemented with 10% fetal calf serum (FCS), 5× 10 − 5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ ml streptomycin. Murine CTL culture medium was composed of MEM-a supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 5× 10 − 5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. All media and supplements were purchased from Gibco BRL (Life Technologies, Sweden). Mouse recombinant IL-4 (mrIL-4) was kindly donated by Schering-Plough Research Institute (Kennilworth, NJ, USA). Mouse recombinant GM-CSF (mrGM-CSF) was purchased from ImmunoKontact (Frankfurt am Main, Germany). Human recombinant IL-2 (hrIL-2) (Dupont) and mouse recombinant IL-12 (mrIL-12) (Hoffman-La Roche) were kindly provided by their respective manufacturers.
2.3. De6elopment of dendritic cells from bone marrow All mice were sacrificed by lethal exposure to ether and then subjected to cervical vertebrae dislocation. Femurs, tibia, ulnae and radii were obtained from
wild type or CD4/CD8 − / − mice. Muscle tissue was removed and bones were placed in a 15 ml tube containing 70% alcohol for 1 min. The bones were then serially transferred two times into 15 ml sterile PBS before the epiphyses were cut using scissors. Marrow tissue was expelled from both the dyaphysis and the epiphyses by fluid pressure using a needle and supplemented DC culture medium. The marrow of approximately one femur diluted in 2 ml medium was placed directly into each well of a 24 well cell culture plate (Costar). Then 1000 IU/ml of both mrGM-CSF and mrIL-4 were added and cells were incubated at 37°C, 7.2% CO2. On days 1 and 3 post culture initiation, the contents of each well were divided in half and the volume of each new well was brought to 2 ml with supplemented DC medium and mrGM-CSF+mrIL-4 (1000 IU/ml final each). On day 6–7 the same procedure was repeated for the final time, but using only 500 IU/ml mrGM-CSF and 1000 IU/ml mrIL-4. Overall cell morphology was examined every day by phase contrast microscopy.
2.4. hrIL-2 /mrIL-12 Cultured bone marrow cells For comparative purposes, CD4/CD8 − / − bone marrow cultures stimulated by an hrIL-2+ mrIL-12 combination were also established. Extraction of bone marrow was performed as described above. After overnight incubation (37°C, 7.1% CO2) of the cells in supplemented DC medium, plastic non-adherent bone marrow cells (2× 106/ml) were removed by pipetting and incubated in supplemented DC medium containing 500 IU/ml hrIL-2 (a generous gift from Dupont) and 0.5 ng/ml mrIL-12 (a generous gift from Hoffman-La Roche). On day 3 of culture, the medium was removed by centrifugation, and replaced by 5 ml supplemented DC medium containing fresh hrIL-2 and mrIL-12 as above.
2.5. Flow cytometry Analysis was performed on day 9 of cell culture. Cells (5× 105) were washed in PBS containing 1% FCS and incubated for 20 min on ice with the primary antibody as indicated. The following primary mAbs (10 mg/ml) were used: anti-B7.1 (IG10) and anti-B7.2 (GLI) (purchased from Pharmagen and kindly provided by Dr Hans Gustaf-Ljungren, MTC, Karolinska Institute), anti-ICAM 1 and anti-LFA-1 (donated by Dr Manuel Pattaroyo MTC, Karolinska Institute), anti-IA (donated by Dr Staffan Pauli, Department lmmunology, Stockholm University). An irrelevant primary antibody, anti IgG1 (Dako) was used as a control. Washed cells (2×) were then incubated on ice for 20 min with either a secondary fluorescein-conjugated anti-mouse IgG F(ab)2, at a
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1:50 dilution, or streptavidin-FITC, as directed by the manufacturer (DAKOPATTS, AB) and washed again. In all cases, washing solution was composed of PBS containing 1% FCS. For each sample, 10 000 events were acquired using a FACScan (Becton Dickinson) and setting forward and side scatter gating to exclude cell debris.
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was calculated according to the formula: % specific release=((experimental release− spontaneous release)/ (maximum release− spontaneous release))× 100.
3. Results
3.1. De6elopment of dendritic cells 2.6. Generation and culture of 6irus-specific CTL To generate a LCMV (lymphocytic choriomeningitis virus) GP 33-41-specific CTL line, the synthetic peptide corresponding to the LCMV GP 33-41 epitope was synthesized by a solid phase method using F-moc solid phase chemistry, and analyzed by reverse phase HPLC and electrospray mass spectrometry. Wild-type C57Bl/6 mice were inoculated i.p., with 102 pfu LCMV. Two weeks after immunization, 25× 106 immune spleen cells were cocultured with 25× 106 irradiated C57Bl/6 spleen cells in the presence of 0.1 mM LCMV GP 33-41 peptide in 12 ml supplemented CTL medium (37°C, 5% CO2). Every 10 days, the CTL line was restimulated with 10 × 106 irradiated and peptide-coated splenocytes in supplemented CTL medium containing 20 IU/ml IL-2. These cells were then used as effectors in cytolytic assays against CD4/ CD8 − / − DC or RMA-S cells loaded or not with the GP 33-41 peptide.
2.7. Immunization using peptide-coated DC DC from C57Bl/6 or CD4/CD8 − / − mice were incubated at 37°C with 30 mM LCMV GP 33-41 peptide for 2 h in serum-free DMEM, washed twice, and injected subcutaneously [13]. Ten to thirteen days after immunization, 25×106 immune spleen cells were isolated and cocultured with 25×106 irradiated autologous spleen cells in the presence of 0.1 mM peptide in 12 ml supplemented CTL medium at 37°C, 5% CO2. Five days later, these cells were used as effectors in CTL assays against RMA-S cells loaded or not with the GP 33-41 peptide.
2.8. CTL assay CTL activity was measured using a standard 51Crrelease assay. Briefly, RMA-S cells or DC from C57Bl/6 CD4/CD8 − / − mice were used as targets. Target cells were labeled with 100 ml of a 1 mCi/ml 51 Cr solution for 1 h at 37°C. Cells were then coated with titrated amounts of peptide (10 − 3 – 10 − 8 mM) for 2 h. Cells were further washed three times before being incubated (3×103 target cells/well) with various amounts of effector cells (E:T ratios of 45:1, 15:1, 5:1, 2:1) for 4 h at 37°C, 5% CO2. After incubation, released radioactivity was measured and specific lysis
Murine recombinant GM-CSF and IL-4 were added at the inception of in vitro cultures as described in the methods. Following overnight incubation at 37°C, CD4/CD8 − / − bone marrow cells formed a mosaic of small round cells which surrounded occasional clumps of bone marrow tissue beneath small mats of agglutinated red blood cells (Fig. 1A). By day 2, small islets of slightly larger cells among the occasional adherent cells could be observed (Fig. 1B). By day 7, prominent islets of cells were readily observed (Fig. 1C) loosely attached to fibroblast-like cells which adhered to tissue-culture treated wells (Fig. 1D).
3.2. Flow cytometric analysis of cells deri6ed from CD4 /CD8 − / − bone marrow cultures Loosely attached cells from cultures depicted in Fig. 1C were readily obtained by gentle resuspension via pipetting and subjected to immunofluorescent analysis, as presented in the materials and methods. For comparison, bone marrow cells of the same origin, but cultured in the presence of hrIL-2 and mrIL-12 (LAK cells) [14], were treated in parallel with the same combinations of primary and secondary reagents. Relative to the negative control cells, CD4/CD8 − / − bone marrow DC (BMDC) expressed clearly elevated levels of cell-surface markers often associated with differentiated/activated DC, such as IA, CD80, CD86, ICAM-1 and LFA-1 (Fig. 2). Note, the two distinct populations of IA + and CD86 + DC cells. In all cases, but the irrelevant primary antibody used as a control, the expression of these five markers characteristic for differentiated/activated DC cells were higher in DC than in LAK cells.
3.3. CD4 /CD8 − / − DCs are efficient antigen presenting cells The functional characteristics of the CD4/CD8 − / − DC cells were tested by two distinct methods. First, the CD4/CD8 − / − DCs were used as target cells for peptide-specific CTLs: CD4/CD8 − / − DCs and RMAS cells were loaded with the H-2Db restricted LCMV GP33-41 peptide, and tested as target cells for GP3341 specific CTLs in a 51Cr-release cytotoxicity assay (Fig. 3). The results indicated that peptide-pulsed
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CD4/CD8 − / − DCs were lysed just as efficiently as peptide-pulsed RMA-S cells, demonstrating that CD4/ CD8 − / − DCs are efficient antigen presenting cells in vitro. Second, the antigen presenting capacity of CD4/ CD8 − / − DCs in vivo [15] was examined. DCs generated from CD4/CD8 − / − and wild type C57Bl/6 bone marrow cultures were incubated in vitro with the LCMV GP33-41 peptide before being injected s.c. in C57Bl/6 mice, as described in the materials and methods section. Ten to thirteen days after immunization, mice were sacrificed and the isolated splenocytes were restimulated 5 days in vitro with autologous splenocytes and the relevant peptide. Resulting CTL activity was tested in a 51Cr-release cytotoxicity assay against RMA-S cells loaded with the LCMV GP33-41 peptide (Fig. 4). The CTLs from mice immunized with either CD4/CD8 − / − or C57Bl/6 control DCs were both peptide specific, and exhibited a somewhat similar cytolytic activity although that of wild type DC was higher than that of knock-out DC. Together, these results indicate that DCs from CD4/ CD8 − / − bone marrow cultures are functionally competent professional antigen presenting cells: they are both able to induce peptide-specific CTL responses and be recognized by peptide specific CTLs. 4. Discussion In view of the important role DC have been found to have in generating antigenic responses [2,3,6], it is of
particular interest to generate both large quantities and purified populations of DC for both research and therapeutic uses. Previous workers have already developed methods to generate large numbers of DC, but the isolation of purified populations or sub-populations of DC are tedious and not always sufficient to guarantee exclusion of contaminant cells that may influence the results [3,8,16]. Here, we first wanted to determine if the same culture supplements used previously with human DC [9,17] would also be adequate for murine DC development. We found that bone-marrow derived CD4/CD8 − / − DC cells cultured as described here exhibit a number of markers that are found on activated/differentiated DC cells (Fig. 2) [6]. The presence of five of them together (IA, CD80, CD86, ICAM-1 and LFA-1), although not individually specific to DC, together with the characteristic morphological appearance of these cells (Fig. 1), permit us to state that bone marrow cells cultured in the regimen of rmIL-4 and rmGM-CSF described here lead to the development of DC cells. Second, we aimed at generating DC cultures totally free of interfering CD3, CD4 and CD8 lymphocytes. We thus developed a methodology to develop DC from CD4/CD8 − / − mice. CD4/CD8 − / − double-knock-out mice were chosen because the overall numbers of CD3 + T cells among bone marrow cells from CD4/ CD8 − / − double knock-out mice are extremely low [10]. Very low initial numbers of contaminating cells coupled with conditions favoring the outgrowth of DCs allowed for the outgrowth of virtually pure DC cultures
Fig. 1. Phase contrast microscopic analysis of CD4/CD8 − / − C57B1/6 mouse bone marrow cultures stimulated with rmGM-CSF and rmIL-4. A, appearance on day1; B, small islets of cells on day 2; C, large islets of loosely adherent cells on day 7; D, underlying stromal cells cleared of loosely adherent DCs on day 7. Bar =25 mm.
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Fig. 2. Flow cytometric analysis of the 9 day-old loosely adherent subpopulation derived from bone marrow cultures stimulated with rmGM-CSF and rmIL-4. Bold lines, bone marrow-derived DC; normal line, bone marrow-derived LAK cells, dotted lines, bone marrow-derived DC reacted with an irrelevant primary antibody (IgG1) as control. Expression of: A, IA; B, CD 80; C, CD 86; D, ICAM-1; E, LFA-1; F, negative control, irrelevant primary antibody (IgG1).
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Fig. 3. Cytolytic activity of anti-LCMV GP 33-41 CTLs against LCMV GP 33-41-loaded CD4/CD8 − / − (circles) and LCMV GP 33-41-loaded RMA-S cells (inverted triangles), both at an E:T ratio of 20:1 (black) and 4:1 (clear). Loading peptide concentrations are displayed on the X-axis.
without extensive depletion steps or other manipulations. The recognition of CD4/CD8 − / − DCs obtained by our method of culture by CTLs and their ability to mount an adequate CTL response were demonstrated (Figs. 3 and 4), indicating that these DCs are functional. Results of these assays also permit us to advance other hypotheses as to the requirements of DC develop-ment. The DCs generated by our method induced a peptide specific CTL response upon immunization of wild-type C57Bl/6 mice (Fig. 4). This indicates that CD8 + and CD4 + cells are not necessary to support development of functional DCs in the bone marrow cultures and that the CD8 and CD4 gene products are not necessary for proper DC development. Remarkably, the CD4/CD8 − / − DCs incubated with the specific MHC class I restricted peptide were sensitive targets for activated CTLs (Fig. 3). These data suggest that CD4/CD8 − / − DCs are fully competent antigen presenting cells and are able to induce vigorous CTL responses. Kronin et al. [18] suggested that the CD8 + subpopulation of DCs have immuno-regulatory properties. Indeed, his data indicated that CD8 + DCs inhibited proliferation of CD8 + CTLs. We therefore expected the CTL responses induced by CD4/CD8 − / − DCs to be stronger than those induced by mixed populations of wild-type DC. Our results indicate rather that wild type DCs are slightly better at inducing CTL re-
sponses. Our data nevertheless do not exclude an immunoregulatory role for the CD8 + DC-subpopulation. Future studies on the immunobiology of CD4/CD8 − / − DCs are required to shed light on this issue. DC cells isolated from CD4/CD8 − / − cultures are thus of particular interest to study the immune responses independent of helper T cells, the importance of DC cells as APC in general, the mechanisms of CD1-restricted responses and the roles played by CD4 + and CD8 + subpopulations [19,20]. In conclusion, we have developed a simple way to generate pure DC cultures from CD4/CD8 − / − double knockout mouse bone marrow progenitors by supplementing cultures with rmGM-CSF and rmIL-4. The resulting cultures were composed of loosely adherent cells easily separated from the underlying strongly adherent stromal cells by gentle pipetting. These bone marrow-derived loosely adherent cells were found to have the overall morphology of DCs and exhibited cell surface markers characteristic of activated/differentiated DC. Functional assays indicated that these cells, when loaded with a MHC class I restricted peptide epitope, were able to elicit a cellular immune reaction in vivo, and to serve as antigen presenting cells in vitro. Our data and technique should prove helpful in the understanding of DC biology.
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Fig. 4. Ability of DC to elicit a peptide-specific CTL response. DC cells derived from wild type (circles) or CD4/CD8 − / − (squares) C57Bl/6 bone marrow were loaded with the LCMV GP 33-41 peptide before being injected in wild type C57Bl/6 mice. Ten to 13 days later, spleens were removed and splenocytes restimulated with peptide in the presence of autologous irradiated splenocytes. RMA-S cells, loaded (black) or not (clear) with the LCMV GP 33-41 peptide, were then used as target cells in two separate experiments (right and left panels). The E:T ratios are displayed on the X-axis.
Acknowledgements This work was supported by grants from The Swedish Cancer Society, The Cancer Society of Stockholm, and the King Gustaf V jubilee fund. We thank L. Szekely, MD, for the microscope pictures and A. Fulgenzi, B.Sc., for his help in type-writing the manuscript.
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