ISOLATION OF HEMATOPOIETIC DENDRITIC CELLS BY HIGH GRADIENT MAGNETIC CELL SORTING

Abstract: Methods are provided for the rapid isolation of highly purified and functionally intact dendritic cells from a mixed cell population, using colloidal superparamagnetic particles. Dendritic cells are enriched from a blood or lymph sample using a two step high-gradient magnetic cell separation. Lymphocytes, natural killer and monocytic cells are depleted by specific binding to markers present on lymphoid and myeloid cells. In a separate step, dendritic cells are enriched by HGMS. Purified dendritic cells are useful as a source of antigen presenting cells for in vitro analysis, and in immunomodulating therapies, particularly for priming naive T cells.

Description: ISOLATION OF HEMATOPOIETIC DENDRITIC CELLS BY HIGH GRADIENT MAGNETIC CELL SORTING INTRODUCTION Technical Field The field of this invention is the isolation of hematopoietic dendritic cells. Background The role and identity of antigen presenting cells has been the subject of controversy in the past. Both macrophages and dendritic cells (sometimes referred to as lymphoid interdigitating dendritic cells) are now known to present antigen. Macrophages present to activated T cells and B cells. However, T helper cell priming is dependent on antigen presentation by dendritic cells. It appears that dendritic cells pick up and process antigen in the peripheral blood, then travel into the lymph, and finish maturation in the paracortical T cell zone of lymph nodes. Macrophages and dendritic cells at all levels of maturity can present antigen to preactivated T cells, but only mature dendritic cells are able to prime naive T cells. Dendritic cells are derived from hematopoietic stem cells in the bone marrow. Precursor and immature dendritic cells are found in the blood and lymph. The morphologically distinct, fully mature dendritic cells are found in the spleen and lymph node. In keeping with their role in antigen presentation, dendritic cells express high levels of MHC class I and class II proteins. A number of methods have been described for the isolation of dendritic cells from peripheral blood mononuclear cells. A common denominator of these techniques is the requirement for an in vitro culture period of 1-2 days. During this time, dendritic cells acquire a low bouyant density and little or no adherence to plastic, features that aid in their separation from other cells. Recently, procedures have been described to isolate dendritic cells from fresh blood. These methods all require a T cell depletion step, followed by flow cytometric sorting. However, the high technological effort required for FACS has prevented its routine use. FACS sorting is a time consuming and cost 5 intensive procedure. FACS sorting has the additional disadvantage in that it is difficult to sort large numbers of cells, or to sort multiple samples at the same time. An alternative approach to cell sorting has been described, whereby magnetic microparticles coupled to antibodies are used to select for specific o cell types. An improved separation process whereby dendritic cells could be sorted from blood, and which allows multiple, and potentially large, samples to be run on the bench would provide numerous benefits in the characterization and use of antigen presenting dendritic cells. 5 Relevant Literature The isolation of dendritic cells after a period of time in culture is described in Cameron et al. (1992) Science 257:383: Langhoff et al. (1991 ) P.N.A.S. 88:7998; Chehimi et al. (1993) J. Gen. Virol. 74:1277; Cameron et al. (1992) Clin. EXD. Immunol. 88:226: Thomas et al. M993. J. Immunol. 150:821 : 0 and Karhumaki et al. (1993) Clin. Exp. Immunol. 91:482. The isolation of dendritic cells from peripheral blood by flow cytometric sorting is described by Thomas et al. (1994) J. Immunol. 153:4016: Ferbas et al. (1994. J. Immunol. 152:4649: and O'Doherty et al. (1994. Immunology 82:487. 5 Activation of naive T cells by antigen pulsed dendritic cells is described in Flamand et al. (1994) Eur. J. Immunol. 24:605-610: Mehta-Damani et al. (1994. J. Immunology 153:996 and Somasse et al. M992> J. EXD. Med. 175:15- 21. High gradient magnetic cell sorting is described in Miltenyi et al. (1990) 0 Cvtometrv 11 :231-238. Molday, U.S. 4,452,773 describes the preparation of magnetic iron-dextran microspheres and provides a summary describing the various means of preparation of particles suitable for attachment to biological materials. A description of polymeric coatings for magnetic particles used in HGMS are found in DE 3720844 (Miltenyi) and Miltenyi et al., U.S. 5,385,707. 5 Methods to prepare superparamagnetic particles are described in U.S. Patent No. 4,770,183. SUMMARY OF THE INVENTION Methods are provided for the rapid isolation of highly purified and functionally intact dendritic cells from a mixed cell population, using colloidal superparamagnetic particles. Dendritic cells are enriched from a blood or lymph sample using a two step high-gradient magnetic cell separation. B- cells, T cells, NK cells and monocytic cells are depleted by specific binding to markers present on lymphoid and myeloid cells. In a separate step, dendritic cells are enriched by HGMS. The isolated dendritic cells are optionally cultured in vitro in the presence of cytokines. Purified dendritic cells are useful as a source of antigen presenting cells for in vitro analysis, and for use in immunomodulating therapy, particularly for priming naive T cells. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A(i) to 1 C(i) show the characterization of peripheral blood mononuclear cells for HLA-DR and CD3, CD14, CD16 and CD19 expression during the separation of dendritic cells. Figures 1A(ii) to 1 C(ii) show the side and forward scatter of cell populations during the separation procedure. Figures 2A and 2B show the expression of CD4 and CD11 c, respectively, by isolated dendritic cells enriched for HLA-DR positive cells. Figures 2C and 2D show the expression of CD33 and CD11b, respectively, in unseparated peripheral blood mononuclear cells and in isolated dendritic cells enriched for HLA-DR positive cells. Figure 3 shows the forward and side scatter of isolated peripheral blood dendritic cells after being subjected to in vitro culture. Figures 4A(i) to 4C(i) show the characterization of peripheral blood mononuclear cells for CD4 and CD3, CD14 and CD16 expression during the separation of dendritic cells. Figures 4A(ii) to 4C(ii) show the side and forward scatter of cell populations during the separation procedure. Figure 5 shows the expression of HLA-DR by isolated dendritic cells enriched for CD4 positive cells. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods for the separation, culture and use of hematopoietic dendritic cells are provided. A blood sample is drawn from a suitable host, and preparation made of mononuclear cells from the blood. Dendritic cells are enriched from the mixed blood cell population by a combination process involving depletion of non-dendritic cells and enrichment of dendritic cells, using high-gradient magnetic cell separation, or a combination of high and low gradient magnetic separation. A suspension of blood cells are labeled with superparamagnetic particles specific for cell surface antigens, then sorted by binding to magnetic columns. The use of high-gradient magnetic cell sorting to enrich for dendritic cells provides several benefits when compared to flow cytometry methods presently used today. The subject methods require inexpensive reagents and apparatus, which are easily used and maintained. By setting up multiple columns, many samples can be processed at the same time. An automated system can be used to simplify processing of large sample numbers and large volumes. The subject methods provide for a highly enriched population of hematopoietic dendritic cells and precursors thereof, usually at least about 90% of the population will be dendritic cells or precursors thereof, more usually at least about 95%. The purity may be evaluated by various methods. Conveniently, flow cytometry may be used in conjunction with light detectable reagents specific for cell surface markers expressed by dendritic cells. The dendritic cells separated by the subject methods are hematopoietic cells characterized as expressing class I and class II MHC proteins, e.g. the human class II proteins HLA-DP, HLA-DQ and HLA-DR; and class I proteins HLA-A, HLA-B and HLA-C. The dendritic cells also express CD45, CD33 and, for the most part, CD4. The cells lack expression of most lymphoid and monocytic specific cell markers, e.g. CD3, CD11 b, CD14, CD16 and CD19. The mature subset of dendritic cells found in blood are characterized by expression of CD11c, high levels of CD33, and CD45RO, and are able to present antigen so as to stimulate naive or preactivated T cells. Precursor dendritic cells are CD11 c negative, express low levels of CD33, are CD45RA positive, and will differentiate into the mature cells after in vitro culture, as described in some detail below. The term dendritic cells (DC) shall be intended to include both mature and precursor cells as found in the blood, unless specifically stated otherwise. Blood sample, as used herein, shall be intended to include hematopoietic biological samples such as blood, lymph, leukophoresis product, bone marrow and the like; also included in the term are derivatives and fractions of such fluids. The sample may be subjected to prior treatment, such as dilution in buffered medium, concentration, filtration, or other gross treatment that will not involve any specific separation. The blood sample is drawn from any site, conveniently by venipuncture. The sample is usually at least about 20 ml, more usually at least about 40 ml and may be as large as about 500 ml, more usually not more than about 250 ml. The blood is treated by conventional methods to prevent clotting, such as the addition of EDTA, heparin or acid-citrate-dextrose solution. A preparation of nucleated cells is made from the sample. Any procedure that can separate nucleated cells from erythrocytes is acceptable. The use of Ficoll-Paque density gradients or elutriation is well documented in the literature. Alternatively, the blood cells may be resuspended in a solution which selectively lyses adult erythrocytes, e.g. ammonium chloride-potassium; ammonium oxalate, etc. The sample of nucleated peripheral blood cells (NPBC) is selectively depleted of non-dendritic cells. Depletion reagents attached to superparamagnetic particles are bound to cell surface antigens that are present on lymphoid and monocytic blood cells, but are low or absent on dendritic cells. Especially useful depletion reagents are antibodies against cell surface antigens. Whole antibodies may be used, or fragments, e.g., Fab, F(ab')2. light or heavy chain fragments, etc. Such antibodies may be polyclonal or monoclonal and are generally commercially available or alternatively, readily produced by techniques known to those skilled in the art. Antibodies selected for use in depletion will have a low level of non-specific staining, and will usually have an affinity of at least about 100 μM for the antigen. Generally, a cocktail of depletion reagents will be used, in order to deplete a wide range of blood cell types. Generally, at least about 75% of the mononuclear peripheral blood cells will be bound by the cocktail of depletion reagents. Suitable antigens for depletion are antigens specific for monocytes, T cells, NK cells and B cells, e.g. CD14 or CD11 b, which is found on monocytes; CD3, which is found on T cells; CD16, which is found on NK cells; and CD19, which is found on B cells. Other useful cell surface antigens include the T cell markers CD2, CD5, CD6 and CD7, the B cell markers CD20, CD21 , CD22, CD23, CD24 and CD37, the NK cell and neutrophil marker CD16, also CD56, CD57 and CD94, and the granulocyte marker CD15. In a preferred embodiment, a cocktail of antibodies specific for CD3, CD14 or CD11 b is used, optionally including CD16 and/or CD19. An alternative combination is antibodies specific for CD3, CD11 b and CD16. The depletion reagent antibodies are coupled to superparamagnetic particles, which can be prepared as described in U.S. Patent nos. 4,452,773 and 4,230,685. The microparticles will usually be less than about 100 nm in diameter, and usually will be greater than about 10 nm in diameter. The exact method for coupling is not critical to the practice of the invention, and a number of alternatives are known in the art. Direct coupling attaches the antibodies to the particles, as described in co-pending patent application no. 08/252,112, herein incorporated by reference. Indirect coupling can be accomplished by several methods. The depletion reagent antibodies may be coupled to one member of a high affinity binding system, e.g. biotin, and the particles attached to the other member, e.g. avidin. One may also use second stage antibodies which recognize species-specific epitopes of the depletion antibodies, e.g. anti-mouse Ig, anti-rat Ig, etc. Indirect coupling methods allow the use of a single magnetically coupled entity, e.g. antibody, avidin, etc., with a variety of depletion antibodies. One preferred method uses hapten-specific second stage antibodies coupled to the superparamagnetic particles, as described in co-pending patent application no. 08/252,112. The hapten specific antibodies will usually have an affinity of at least about 100 μM for the hapten. The depletion antibodies are conjugated to the appropriate hapten. Suitable haptens include digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl, etc. Methods for conjugation of the hapten to antibody are known in the art. While not necessary for practice of the subject methods, it may be useful to label the depletion antibodies with a fluorochrome, e.g. phycoerythrin, FITC, rhodamine, Texas red, allophycocyanin, etc. The fluorochrome label may be used to monitor microscopically or by flow cytometry the cell composition after the depletion and enrichment steps. Fluorescent labeling may conveniently utilize the same indirect coupling system as the magnetic particles. For example, a cocktail of digoxigenin-coupled depletion antibodies may be used in combination with anti-digoxigenin antibody coupled to magnetic particles, followed by labeling with a fluorochrome conjugated antibody directed to the anti-hapten antibody. The depletion reagent antibodies are added to a suspension of NPBC, and incubated for a period of time sufficient to bind the available cell surface antigens. The incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, so that the efficiency of the magnetic separation is not limited by lack of antibody. The appropriate concentration is determined by titration. The medium in which the cells are separated will be any medium which maintains the viability of the cells. A preferred medium is phosphate buffered saline containing from 0.1 to 0.5% BSA. Various media are commercially available and may be used according to the nature of the cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc. Where a second stage magnetically coupled antibody is used, the cell suspension may be washed and resuspended in medium as described above prior to incubation with the second stage antibodies. Alternatively, the second stage antibody may be added directly into the reaction mix. When directly coupled depletion antibodies are used, the cell suspension may be used directly in the next step, or washed and resuspended in medium. The suspension of magnetically labeled cells is applied to a column or chamber as described in WO 90/07380, herein incorporated by reference. The matrix may consist of closely packed ferromagnetic spheres, steel wool, wires, magnetically responsive fine particles, etc. The matrix is composed of a ferromagnetic material, e.g. iron, steel, etc. and may be coated with an impermeable coating to prevent the contact of cells with metal. The matrix should have adequate surface area to create sufficient magnetic field gradients in the separation chamber to permit efficient retention of magnetically labeled cells. The volume necessary for a given separation may be empirically determined, and will vary with the cell size, antigen density on the cell surface, cell number, antibody affinity, etc. In order to maximize the purity of the final cell preparation, a two stringency system is employed, where the depletion step captures a high percentage of labeled cells and the enrichment step captures a lower percentage of labeled cells. This reduces the probability that labeled cells will be carried over from the first separation step into the second. The stringency of the depletion column will be such that at least about 95% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 99% of the labeled cells will be retained, and preferably at least about 99.9% retained. The geometry, matrix composition, magnetic field strength, size and flow rate of the ferromagnetic column will determine the percent of labeled cells that are retained on the column. Factors that will increase the stringency are increased column size and length, decreased flow rate, and a finer matrix composition. A column matrix of fibers is preferred for the depletion step. An empirical determination of the stringency may be made by analysis of bound and unbound cells. The labeled cells are bound to the matrix in the presence of a magnetic field, usually at least about 100 mT, more usually at about 500 mT, usually not more than about 2T, more usually not more than about 1T. The source of the magnetic field may be a permanent or electromagnet. The unbound cells contained in the eluate are collected as the eluate passes through the column. For greater purity, the unbound cells may be passed a second time over the magnetic column. The unbound cells are used in an enrichment step, to select for dendritic nucleated cells. Enrichment reagents attached to superparamagnetic particles are bound to cell surface antigens that are present on dendritic cells. Of particular interest is the use of reagents specific for MHC class II proteins, e.g. HLA-DR, HLA-DQ and HLA-DP or other cell surface markers specifically present on dendritic cells, such as CD4 for dendritic cells and precursors, or as appropriate, CD11c for mature dendritic cells. CD45RO may be used to select for mature dendritic cells, and CD45RA to select for precursor cells. The choice of enrichment reagent will determine to some extent the choice of depletion reagents, based on the distribution of expression of the particular markers. The depletion reagents will be selected so as to specifically deplete non-dendritic cells expressing the enrichment marker. For example, CD4 is highly expressed by T cells and monocytes, and so the depletion step preceding CD4 selection will include T cell and monocyte specific reagents. MHC class II proteins are absent or expressed at low density on T cells, but are expressed by B cells and monocytic cells, and so the depletion step preceding HLA class II selection will include B cell and monocytic cell specific reagents. Reagents specific for the HLA class II proteins may be allele specific, i.e. directed to polymorphic regions of the protein, or directed to conserved sequences. HLA-DRα is relatively invariant in sequence, while HLA-DRβ is highly polymorphic. Generally, reagents will be chosen that recognize HLA proteins from a large number of individuals. However, it may be desirable to isolate dendritic cells having a particular haplotype through the use of allele specific reagents. Conveniently, the enrichment reagent will provide for magnetic labeling through an indirect coupling different from that used for the depletion. The initial binding reaction may combine both enrichment and depletion reagents. The second stage magnetic particles specific for the enrichment reagent is added after completion of the depletion. Alternatively, a directly coupled enrichment reagent may be used. The enrichment reagents, superparamagnetic particles, columns and buffers are prepared as described for the depletion reagents, however, the stringency for the enrichment column will be lower than for the depletion column. The stringency of the enrichment column will be such that at least about 50% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 80% of the labeled cells will be retained, usually not more than 95% retained. A column matrix of spheres is preferred for the enrichment step. The cells are bound to the magnetic matrix. After the initial binding, the matrix is washed with any suitable physiological buffer to remove unbound cells. The unbound cells are discarded. The bound cells are released by removing the magnetic field, and eluting in a suitable buffer. The cells may be collected in any appropriate medium which maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, PBS-EDTA, PBS. Iscove's medium, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc. In many cases the separation procedure will perform the depletion step first, followed by the enrichment step. If the enrichment step is to be performed first, then an additional step is necessary after the enrichment, in order to remove the magnetic label from the enriched cells. This may be accomplished by any suitable method. The enriched cell population may be incubated with a solution of dextranase, where the dextranase is present at a concentration sufficient to remove substantially all microparticles from the labeled cells. Usually the reaction will be complete in at least about 15 minutes. The depletion step may then be performed as previously described with the dextranase treated cells. Alternatively, the enrichment step may be performed first, and the depletion step modified to use large magnetic spheres in place of the microparticles. The use of such magnetic spheres has been previously described, and the reagents are commercially available. The enriched cell population is incubated with highly magnetic polymer spheres of about 1 to 10 μm diameter conjugated to the depletion antibody cocktail. The mixture of cells is then placed in close proximity to a magnetic field. Substantially all cells bound to the polymer spheres are bound to the magnet within about 1 minute, and not more than about 5 minutes. The unbound cells may be decanted and used. After the depletion and enrichment steps are complete, the cells may be used immediately for antigen presentation, further analysis of DC function, as a source of mRNA for use in cDNA synthesis, etc. The mature cells and progenitor cells may be compared by cDNA subtraction to determine differences in gene expression during the maturation process, and to identify specific genes expressed by mature dendritic cells. Alternatively, the cells may be cultured in vitro for a period of time sufficient to induce further maturation, usually at least about 1 day and not more than about 7 days, more usually about 2 to 3 days. The dendritic cells are cultured in an appropriate liquid nutrient medium, which medium may further comprise one or a combination of cytokines at a concentration sufficient to enhance the differentiation of precursor dendritic cells into mature antigen presenting cells. Cells will be grown at a concentration from about 10 4 per ml to about 10 6 per ml, usually about 10 4 to 10 5 . Various media are commercially available and may be used, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's medium, etc. , frequently supplemented with serum, usually heat inactivated normal human serum, generally at a concentration of from about 5-15%, preferably about 10%. Appropriate antibiotics to prevent bacterial growth and other additives, such as pyruvate (0.1-5 mM), glutamine (0.5 - 5 mM), 2-mercaptoethanol (1 - 10 x 10 -5 M) may also be included. Cytokines of interest include IL-1 , IL-2, IL-3, IL-4, GM-CSF and TNF-α. The addition of GM-CSF to cultures of precursor, i.e. CD11c negative, dendritic cells is of particular interest. The factors that are employed may be naturally occurring or synthetic, e.g. prepared recombinantly, and may be human or of other species, e.g. murine, preferably human. Alternatively, monocyte conditioned medium may be used as a source of cytokines (see for example, O'Doherty et al. (1994) supra.). The amount of the cytokines will generally be in the range of about 1 ng/ml to 1 μg/ml. Appropriate culture conditions may be empirically tested by assaying the resulting cells for their ability to present antigen. Various methods of determining antigen presenting activity are known in the art and may be used for this purpose. For example, the ability of cells to induce proliferation of freshly isolated T cells stimulated with sub-optimal concentrations of anti-CD3 may be measured (see Thomas et al. (1994), supra.). In most cases, increased ability to present antigen is found after culture, frequently accompanied by an increase in the expression of B7 (CD80, CD86). The enriched population of dendritic cells, either before or after culture, may be used as antigen presenting cells to prime T cells in vivo or in vitro. The cells are combined with a protein antigen, or with a peptide thereof. Antigenic peptides will usually be from about 6 to 20 amino acids in length, more usually from about 10 to 18 amino acids. The peptides may have a sequence derived from a wide variety of proteins. In many cases it will be desirable to use peptides which act as T cell epitopes, usually immunodominant sequences. The epitopic sequences from a number of antigens are known in the art. Alternatively, the epitopic sequence may be empirically determined, by isolating and sequencing peptides bound to native MHC proteins, by synthesis of a series of peptides from the target sequence, then assaying for T cell reactivity to the different peptides, or by producing a series of binding complexes with different peptides and quantitating the T cell binding. Preparation of fragments, identifying sequences, and identifying the minimal sequence is amply described in U.S. Patent No. 5,019,384, iss. 5-28-91 , and references cited therein. The peptides may be prepared in a variety of ways as known in the art. Antigens of interest include tumor cell antigens, allogeneic MHC antigens, allergens, proteins of pathogenic organisms, including viruses, e.g. HIV-1 , hepatitis, herpesviruses, enteric viruses, respiratory viruses, rhabdovirus, rubeola, poxvirus, paramyxovirus, morbillivirus, filovirus, ere. Infectious agents of interest also include bacteria, such as Pneumococcus, Staphylococcus, Bacillus, Streptococcus, Meningococcus, Gonococcus, Eschericia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Yersinia, Listeria, Corynebactehum, Vibrio, Clostridia, Chlamydia, Mycobacterium, Helicobacter and Treponema; protozoan pathogens, and the like. Dendritic cells are pulsed in vitro with antigens by placing the cells in a physiologically acceptable buffer containing antigen at a concentration from at least about 0.1 μM to as much as about 1 mM. Peptide antigens will typically be effective at a lower concentration than intact protein antigens, or cell lysates. The cells will be incubated with antigen, generally at 37° C, for a period of time sufficient to bind the antigen to the cell. Peptide antigens will usually be incubated for at least about 1 hour, and for as long as 6 hours or more. Intact protein antigens will usually be incubated for at least about 3 hours, and for as long as about 12 hours or more. The antigen pulsed DCs may be used to stimulate a T cell response against the antigen. The T cells may be in vivo, either an autologous or allogeneic host, or may be an in vitro culture. For in vivo use the dendritic cells may be administered in any physiologically acceptable medium, by sub¬ cutaneous, intravenous, intra-dermal, etc. administration. Usually, at least 1x10 4 cells will be administered, preferably 1x10 5 or more. The cells may be introduced by injection, catheter, or the like. If desired, depending upon the purpose of the introduction of the cells, factors may also be included, such as the interleukins, e.g. IL-2 and IL-4, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g. γ-interferon, erythropoietin, etc. The amount of these various factors will depend upon the purpose of the administration of the cells, the particular needs of the patient, and will normally be determined empirically. The antigen pulsed dendritic cells or membranes thereof may be used as immunoadsorbants to obtain antigen specific T cells. Quantitation of T cells may be performed to monitor the progression of a number of conditions associated with T cell activation, including autoimmune diseases, graft rejection, viral infection, bacterial and protozoan infection. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Example 1 Purification of CD3-. CD14-. CD16-. CD19-. HLA-DR+ Dendritic Cells bv HGMS Materials and Methods Preparation of human NPBC. NPBC were obtained from leukocyte-rich buffy coats by centrifugation over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). After centrifugation, interphase cells were collected, resuspended in buffer and sedimented at 300 x g and then once again resuspended in buffer and centrifuged at 200 x g to remove platelets. Labeling. About 2 x 10 8 NPBC were incubated with biotinylated α-CD3 (5 μg/ml), α-CD14 (2 μg/ml), α-CD16 (2 μg/ml), α-CD19 (5 μg/ml), digoxigenin- conjugated anti-HLA-DR monoclonal antibody (mAb) (5 μg/ml) and human IgG (1.6 mg/ml) for 10 minutes at 8°C in a volume of 1 ml PBS, 1 % bovine serum albumin, and then washed twice (300 x g). Magnetic Labeling. Cells labeled with biotinylated antibodies as described above were incubated with streptavidin-conjugated colloidal superparamagnetic microparticles at 8°C in a volume of 1 ml. After 15 minutes, streptavidin-CyChrome (Pharmingen, San Diego, CA) was added (2 μg/ml) and the cells were incubated for another 5 minutes at 8°C. The cells were then washed again and resuspended in 1 ml of buffer. Depletion of Cells by HGMS. Magnetically labeled CD3, CD14, CD16 and CD19 positive cells were removed by passage over a CS column (magnetizable steelwool matrix) inserted in a MACS permanent magnet and sorted essentially as described by Miltenyi et al. (1992) supra. For optimal depletion, the flow rate was kept to approximately 3 ml/min. by using a 22G needle at the outlet of the MACS column. The cells were then washed off with 1 ml of buffer. To evaluate the efficiency of the cell separation, aliquots of unseparated cells, and from the magnetic and nonmagnetic cell fractions were analyzed by two parameter cytometric analysis with a FACScan flow cytometer (Becton Dickinson, San Jose, CA). 10,000 events per sample were recorded and analyzed using FACScan research software (Becton Dickinson). Live cells were gated according to scatter and relative propidium iodide to phycoerythrin staining, with propidium iodide (PI) specifically staining for dead cells. Anti-digoxigenin Labeling. NPBC depleted of CD3, CD14, CD16 and CD19 positive cells were incubated with anti-digoxigenin monoclonal antibody conjugated to colloidal superparamagnetic microparticles at 8°C in a volume of 1 ml. After 15 minutes, anti-digoxigenin monoclonal antibodies conjugated to PE were added, and the cells were incubated for an additional 5 minutes at 8oC. The cells were then washed again, and resuspended in 0.5 ml of buffer. Enrichment of Cells by HGMS. Magnetically labeled HLA-DR+ cells were enriched on a MiniMACS column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) inserted in a MiniMACS permanent magnet. Negative cells were washed off the column at flow rates of approx. 0.35 ml/min. in a volume of 1 ml. The column was then washed again four times with 0.5 ml of buffer. Finally, the HLA-DR + cells were eluted from the column outside of the magnet in 1 ml of PBS/BSA. The number of live cells was evaluated just before and after loading the MACS column in the depletion and enrichment step by a Neubauer counter chamber. Dead cells were excluded by trypan blue staining. To evaluate the efficiency of the cell separation, aliquots of unseparated cells, and from the magnetic and non-magnetic cells fractions were analyzed by flow cytometry using a FACScan. Counterstaining. Freshly isolated dendritic cells from peripheral blood were incubated with either α-CD4-FITC, α-CD11 c-FITC, α-CD33-FITC or α- HLA-DR for 10 minutes at 8° C. Afterwards, cells were washed once and then analyzed by flow cytometry. Dendritic Cell Culture. Dendritic cells were cultured in complete RPMI 1640 medium containing serum, penicillin, streptomycin, glutamine and 5% FCS for 70 hours. Results The analysis of the sorted populations is shown in Figure 1. A dendritic cell population was isolated with 95% purity, having the phenotype of CD3", CD 14-, CD16-, CD19", HLA-DR + . Dendritic cells were CD4 positive. Two different populations were present, and could be distinguished on the basis of CD11 c expression. Dendritic cell precursors are CD11c negative, and mature dendritic cells are CD11 c positive. Freshly isolated dendritic cells lack the characteristic dendritic morphology, and have the appearance of medium sized cells with a slightly higher forward scatter than lymphocytes. Figures 1A to 1C show the results of flow cytometric monitoring of the magnetic purification procedure. CD3", CD14-, CD16", CD19" HLA-DR + DC were enriched from fresh NPBC by immunomagnetic depletion of CD3, CD14, CD16 and CD19 positive cells and subsequent enrichment of HLA-DR positive cells. CD3, CD14, CD16 and CD19 positive cells were indirectly magnetically labeled using biotinylated monoclonal antibodies and streptavidin-conjugated colloidal superparamagnetic microparticles. HLA-DR positive cells were indirectly labeled using a digoxigenin coupled anti-HLA-DR mAb and digoxigenin mAb coupled to colloidal superparamagnetic particles. Cells were stained for CD3, CD14, CD16 and CD19 using streptavidin CyChrome, and for HLA-DR using anti-digoxigenin mAb coupled to phycoerythrin. Live cells were gated according to light scatter signals. The percentage of dendritic cells is shown beside the boxed cells. Figure 1A(i) is a dotplot of CyChrome anti-CD3; CD14; CD16 and CD19 staining vs. PE anti-HLA-DR staining of unseparated NPBC. Figure 1A(ii) is a dotplot of forward scatter (FSH-H) and side scatter (SSH-H) signals from the same population as (i). Figure 1 B(i) shows NPBC after magnetic depletion of CD3, CD14, CD16 and CD19 positive cells. Figure 1B(ii) is the forward and side scatter from the same population as (i). Figure 1C(i) shows NPBC after depletion of CD3, CD14, CD16 and CD19 positive cells and enrichment for HLA-DR+ cells. Figure 1 C(ii) is the forward and side scatter from the same population as (i). Figure 2A is a dotplot of CD3- CD14- CD16- CD19- HLA-DR + dendritic cells after counterstaining with CD4-FITC. Figure 2B shows the same population as (2A) after counterstaining with CD11c-FITC. Figure 2C shows a comparison of CD33 staining for unseparated peripheral blood mononuclear cells and for the isolated dendritic cell population shown in (2A). Among unseparated cells, CD33 is expressed on monocytic cells. Expression of CD33 on dendritic cell precursors is weaker than that seen with monocytes; with mature dendritic cells it is stronger. Figure 2D shows a comparison of CD11b staining for unseparated peripheral blood mononuclear cells and for the isolated dendritic cell population shown in (2A). Among unseparated peripheral blood mononuclear cells, CD11b is expressed at high levels on monocytes but in lesser amounts on NK cells. Expression on dendritic cells is comparable to NK cells, but weaker than that seen with monocytes. Figure 3 is a dotplot of the scatter signal from CD3- CD14- CD16- CD19" HLA-DR + dendritic cells after a culture period of 70 hours. As compared to the freshly isolated cells, shown in Figure 1 C(ii), there is a significant increase in the side scatter. Freshly isolated dendritic cells lack the characteristic dendritic morphology and have the appearance of medium sized cells with a slightly higher forward scatter than lymphocytes. One can distinguish between round cells with simple oval or inented nuclei and less round cells with mildly ruffled borders and more complicated or lobulated nuclei. After a period of culture, blood DCs develop the typical DC morphology, exhibiting dendritic processes. Example 2 Purification of CD3-. CD14-. CD16- CD4 + Dendritic Cells bv HGMS The materials and methods were essentially as described for Example 1 , with the following differences. 2 x 10 8 freshly isolated peripheral blood mononuclear cells were incubated with biotinylated α-CD3 (5 μg/ml), α-CD14 (2 μg/ml) and α-CD16 (2 μg/ml); and digoxigenin-conjugated α-CD4 mAb (2 μg/ml) and human IgG (1.6 mg/ml) for 10 minutes at 8°C in a volume of 1 ml, and then washed twice (300 x g). The subsequent immunomagnetic depletion step selected for cells lacking CD3, CD14 and CD16. The enrichment step selected for cells expressing CD4. The analysis of the sorted populations is shown in Figure 4. A dendritic cell population was isolated with 97% purity, having the phenotype of CD3 * , CD14-, CD16" and CD4 + . Figures 4A to 4C show the results of flow cytometric monitoring of the magnetic purification procedure. CD3-, CD14-, CD16 * and CD4 + DC were enriched from fresh NPBC by immunomagnetic depletion of CD3, CD14 and CD16 positive cells and subsequent enrichment of CD4 positive cells. CD3, CD14 and CD16 positive cells were indirectly magnetically labeled using biotinylated monoclonal antibodies and streptavidin-conjugated colloidal superparamagnetic microparticles. CD4 positive cells were indirectly labeled using a digoxigenin coupled anti-CD4 mAb and digoxigenin mAb coupled to colloidal superparamagnetic particles. Cells were stained for CD3, CD14 and CD16 using streptavidin CyChrome, and for CD4 using anti- digoxigenin mAb coupled to phycoerythrin. Live cells were gated according to light scatter signals. The percentage of dendritic cells is shown beside the boxed cells. Figure 4A(i) is a dotplot of CyChrome anti-CD3; CD14 and CD16 staining vs. PE anti-CD4 staining of unseparated NPBC. Figure 4A(ii) is a dotplot of forward scatter (FSH-H) and side scatter (SSH-H) signals from the same population as (i). Figure 4B(i) shows NPBC after magnetic depletion of CD3, CD14 and CD16 positive cells. Figure 4B(ii) is the forward and side scatter from the same population as (i). Figure 4C(i) shows NPBC after depletion of CD3, CD14 and CD16 positive cells and enrichment for CD4+ cells. Figure 4C(ii) is the forward and side scatter from the same population as (i). Figure 5 is a dotplot of CD3- CD14" CD16- CD19- CD4 + dendritic cells after counterstaining with α-HLA-DR. Almost all cells isolated according to expression of CD4 express HLA-DR. It is evident from the above results that the subject invention provides for a simple, fast method for separating hematopoietic dendritic cells and dendritic precursor from blood. The ease of operation, and ability to scale up the number and size of samples, provide significant benefits over existing methods. The cells are useful as a source of antigen presenting cells for in vivo and in vitro T cell stimulation. The cells are also useful in analysis of dendritic cell growth, differentiation and function. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for enrichment of hematopoietic dendritic cells from a blood sample, the method comprising: preparing a suspension of nucleated cells from said blood sample; 5 adding to said suspension of nucleated cells magnetically coupled reagents specific for one or more cell surface antigens expressed by non- dendritic hematopoietic cells and absent on dendritic cells; passing said suspension of cells through a ferromagnetic matrix in the presence of a magnetic field; o collecting cells that are unbound to said ferromagnetic matrix to provide a depleted sample substantially free of cells comprising said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells; adding to said depleted sample magnetically coupled reagent specific 5 for a cell surface antigen expressed by dendritic cells; passing said depleted sample through a ferromagnetic matrix in the presence of a magnetic field; washing said matrix of unbound cells; and eluting bound cells from said matrix in the substantial absence of said 0 magnetic field to provide an enriched cell sample comprising hematopoietic dendritic cells. 2. A method according to Claim 1 , wherein said enriched cell sample comprising hematopoietic dendritic cells comprises at least 95% 5 hematopoietic dendritic cells. 3. A method according to Claim 2, wherein said cell surface antigen expressed by dendritic cells is CD4 or an HLA class II protein. 0 4. A method according to Claim 3, wherein said HLA class II protein is HLA-DR. 5. A method according to Claim 2, wherein said magnetically coupled reagent specific for a cell surface antigen expressed by dendritic cells 5 is specific for a cell surface antigen expressed by mature hematopoietic dendritic cells. 6. A method according to Claim 5, wherein said cell surface antigen expressed by mature hematopoietic dendritic cells is CD33, CD11c or CD45RA. 7. A method according to Claim 2, wherein said magnetically coupled reagent specific for a cell surface antigen expressed by dendritic cells is specific for a cell surface antigen expressed by precursor hematopoietic dendritic cells. 8. A method according to Claim 7, wherein said cell surface antigen expressed by precursor hematopoietic dendritic cells is CD45RO. 9. A method according to Claim 2, wherein said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells are CD3 and one of CD14 and CD11b. 10. A method according to Claim 9, wherein said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells further comprise at least one of CD16 and CD19. 11. A method for enrichment of antigen presenting cells, the method comprising enriching for a population of hematopoietic dendritic cells according to Claim 1 ; adding said enriched population to a liquid culture medium and growing in vitro for at least one day. 12. A method according to Claim 11 , wherein said liquid culture medium comprises the cytokine GM-CSF at a concentration sufficient to enhance the differentiation of precursor hematopoietic dendritic cells to mature dendritic cells. 13. A kit for separation of dendritic cells from blood, comprising: two columns of a ferromagnetic matrix; a cocktail of magnetically coupled antibody specific for CD 14 and CD3; magnetically coupled antibody specific for at least one of CD4 and HLA- DR.

Inventors: MILTENTI STEPHAN, SCHMITZ JUERGEN, MILTENTI, STEPHAN, SCHMITZ, JUERGEN
Applicants: MILTENYI BIOTECH INC, MILTENYI BIOTECH, INC.
Application year: 1996
Main classification: G01N33
IPC codes: G01N33