WO1998013382A1

WO1998013382A1 - A process for culturing cells

Info

Publication number: WO1998013382A1
Application number: PCT/AU1997/000647
Authority: WO
Inventors: Kenneth Douglas Shortman; Dolores Joyous Saunders; Karen LUCAS; Li Wu
Original assignee: Amrad Operations Pty. Ltd.
Priority date: 1996-09-27
Filing date: 1997-09-29
Publication date: 1998-04-02
Also published as: AUPO263696A0

Abstract

The present invention relates generally to a cell culture process and to cells produced therefrom. More particularly, the present invention provides a method of developing dendritic cells from cultured precursor cells. The dendritic cells of the present invention are useful inter alia as adjuvants, immune system modulating agents, anti-cancer agent, immunotherapeutic agents and tolerizing agents for transplantation. The cell culture process of the present invention may also be extrapolated to a method of stimulating development of dendritic cells in vivo.

Description

A PROCESS FOR CULTURING CELLS

The present invention relates generally to a cell culture process and to cells produced therefrom. More particularly, the present invention provides a method of developing dendritic cells from cultured precursor cells. The dendritic cells of the present invention arc useful inter alia as adjuvants, immune system modulating agents, anti- cancer agents, immunotherapeutic agents and tolerizing agents for transplantation. The cell culture process of the present invention may also be extrapolated to a method of stimulating development of dendritic cells in vivo.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Dendritic cells are sparsely but widely distributed migratory cells of bone marrow origin (Steinman, 1991). Their function has been considered lo involve the collection of antigen in various tissue sites, transport of the processed antigen to the T cell areas of the lymphoid tissue, presentation of the anligenic peplides and stimulation of the immune response of T cells (Knight & Slagg, 1993).

Dendritic cells are generally considered lo be of myeloid origin. Support for this view is based on studies of the outgrowth of dendritic cells, in vitro, under Ihe influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Rcid et al. , 1990; Inaba el al. , 1992; Caux et al., 1992). Said dendritic cells appear to derive from a progenitor capable, also, of differentiating to granulocyles and macrophagcs (Inaba et al. , 1993). The myeloid nature of the dendritic cell is further supported by the direct development of dendritic cells from blood monocylcs (Ro n i et al. , 1994; Kabel et al. , 1989; Rossi e. al. , 1992).

The dendritic cells of the thymus (Fairchild & Austy , 1990) arc believed to be the mediators of thymic negative selection, the process of elimination of potentially self reactive cells before mature T cells emmigrale lo the periphery. Thymic dendritic cells are a relatively sparse population (0. 1 % of thymocytcs) wilh a rapid turnover (7-day life span) (Kampinga et ai. , 1990; Wu et al. , 1995). Until the present invention, they were considered lo arrive in the thymus preformed from the blood stream.

Adoptive transfer of highly purified precursor cells isolated from murinc thymus has demonstralcd thai some thymic dendritic cells arise in situ from an intra-thymic precursor and are related lo the lymphoid lineage. Said intra-thymic precursor appears identical to the earliest T cell precursor population in the adult thymus, Ihe "low CD4" precursor. The low CD4 precursor population differs from multi-potent stem cells in its developmental potential in that although it retains the capacity to form B cells and NK cells, as well as T cells, it has lost the capacity to form myeloid or erylhroid cells (Wu el al. , 1991 ; Ardavin et al. , 1993; Wu et al. , 1995). It is therefore a "lymphoid restricted" precursor.

By virtue of their highly developed antigen presenting capacity, Ihe use of dendrilic cells as cellular vectors for anti-tumour and infectious disease vaccines or as induccrs of transplantation tolerance would be most beneficial. However, until the advent of the present invention, thymic precursors and precursors related to the lymphoid lineage could not be cultured in vitro to differentiate lo dendrilic cells.

In working leading up to ihe present invention the inventors have developed an in vitro process for developing dendritic cells from thymic precursor cells and more particularly from low CD4 precursor cells and pro-T cells. Accordingly, one aspect of Ihe present invention contemplates a method of developing dendritic cells from precursor cells, said method comprising culluring said precursor cells in the presence of an effective amount of a mixture of at least three cylokines or functional derivatives thereof wherein al lcasl two cylokines are TNFα and IL-1.

More particularly the present invention contemplates a method of developing dendritic cells from precursor cells, said method comprising culturing said precursor cells in the presence of an effective amount of a mixture of at least three cytokines or functional derivatives thereof wherein at least three cytokines are TNFα, IL- 1 and IL-3

Reference hereinafter to a specific cytokine should be read as including reference to all forms of said cytokine and functional derivatives thereof For example, reference to IL-1 should be understood to include reference to IL-1 β and IL-lα and functional derivatives thereof.

Preferably, IL-1 is IL-1 β.

Reference hereinafter lo "dendrilic cells" should be read as including reference lo cells exhibiting dendrilic cell phcnotype or functional activity and mutants or variants thereof. "Variants" include, but are not limited lo, cells exhibiting some but nol all of the phenotypic features or functional aclivities of dendrilic cells. Said phenolypic features may include expression of one or more of MHCII, CDl lc, CD44, DEC-205, CD80 or CD86. Said functional activity includes, but is not limited to, the ability to stimulate the proliferation of allogeneic CD4 T cells in mixed leukocyte cultures. "Mutants" include, but are not limited to dendritic cells which are transgcncic wherein said transgeneic cells are engineered to express one or more genes encoding antigens, immune modulating agents, receptors or cylokines.

The term "precursor cells" should be understood lo refer to cells which are not fully differentiated and which are derived from haemalolymphoid tissue. Examples include, but are not limited to, precursor cells derived from bone marrow, spleen, lymph node, Ihymus or blood. Said precursor cells are exemplified by but not limited to mulli- polenlial stem cells, myeloid precursor cells or lymphoid precursor cells such as Lineage bone marrow derived precursors, CD4¹" Ihymocytes, neonatal murine CD4 CD8 Ihymocytes and human CD34 CD4 CD8 Ihymocytes. Some or all of the precursor cells may also be transgenic in that said cells may be engineered to express one or more genes such as genes encoding antigens, immune modulating agents, receptors or cylokines. Preferably said precursor cells arc lymphoid precursor cells.

Reference to the use of a "mixture" of cylokines according lo the method of the present invention should be understood lo encompass both the administration of a single composition comprising said cytokines or the sequential or simultaneous administration of one or more of said cylokines.

According to this preferred embodiment, the present invention provides a method of developing dendritic cells from lymphoid precursor cells, said method comprising culturing said precursor cells in Ihe presence of a mixture of at least three cytokines or functional derivatives thereof wherein al least two cytokines are TNFα and IL-1.

More particularly the present invention provides a method of developing dendritic cells from lymphoid precursor cells said method comprising culturing said precursor cells in Ihe presence of a mixture of al least three cytokines or functional derivatives thereof wherein at least three cylokines are TNFα, IL- 1 and IL-3.

Even more particularly said IL- 1 is IL-lβ.

Preferably said lymphoid precursor cells are T cell precursor cells and even more preferably low CD4 precursor cells or pro-T cells derived from adult mouse thymus. Said low CD4 precursor cell is defined on the basis o the phenotypic profile CD4'", CD8-, CD3-, CD25 + , CD45 + c-kit-l- while the pro-T cell is more differentiated than the low CD4 cell and therefore a downstream T cell precursor population defined on the basis of phenotypic profile CD4-, CD8-, CD3-, CD25+, CD45+c-kit+.

Accordingly in a most preferred embodiment the present invention contemplates a method of developing dendritic cells from low CD4 precursor cells, said method comprising culturing said precursor cells in the presence of a mixture of least three cytokines or functional derivatives thereof wherein at least two cytokines are TNFα and IL-1 .

More particularly in a most preferred embodiment the present invention contemplates a method of developing dendritic cells from low CD4 precursor cells, said method comprising culturing said precursor cells in the presence of a mixture of at least three cylokines or functional derivatives thereof which al least three cytokines arc TNFα, IL- 1 and IL-3.

Most particularly said IL-1 is IL-1 β

In yet another most preferred embodiment the present invention contemplates a method of developing dendritic cells from pro-T cells said method comprising culluring said pro-T cells in the presence of a mixture of least three cytokines or functional derivatives thereof wherein at least two cylokines are TNFα and IL-1 .

More particularly in another most preferred embodiment the present invention contemplates a method of developing dendritic cells from pro-T cells, said method comprising culturing said pro-T cells in the presence of a mixture of least three cytokines or functional derivatives thereof wherein at least three cylokines arc TNFα, IL- 1 and IL-3.

Most particularly said IL-1 is IL-1 β

The present invention extends to any mixture of cytokines which is capable of facilitating development of dendritic cells from precursor cells, in particular from low CD4 precursor cells and pro-T cells, and which contain at least TNFα, and IL-1 . Prelerably, the mixture comprises TNFα, IL- 1 and IL-3 and even more prelerably TNFα, IL- 1 β and IL-3 Most preferably the mixture comprises TNFα, IL-1 β, IL-3 and an effective number of cytokines selected from IL-7, SCF, Flt3L and CD40 ligand (CD40L) or functional derivatives thereof

"Functional derivatives" include fragments, parts, portions, chemical equivalents, mutants, homologs and analogs from natural synthetic or recombinant sources including fusion proteins Chemical equivalents of said cytokines can act as functional analogs of said cytokines Chemical equivalents may not nccessaiily be derived from said cytokines but may share certain conformational similarities Alternatively, chemical equivalents may be specifically designed to mimic certain physiochemical properties of said cytokines Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screenings

Homologs of said cytokines contemplated herein include, but are not limited to, proteins derived from different species

Derivatives may be derived from insertion, deletion or substitution of amino acids Amino acid insertional derivatives include amino and/or caiboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in said cytokines although random insertion is also possible with suitable screening of the resulting product Deletio al variants are characterised by the removal of one or moie amino acids fiom the sequence Substitional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place Additions to amino acid sequences include fusions with other peptides or polypeptides It is possible, for example, that the subject preferred cytokines may be substituted by other cytokines or hybrid cytokines antibodies directed to said cytokine receptor molecules, such as the CD40 antibody FGK45 5 or functional derivatives thereof A hybrid cytokine may comprise a combination of cytokines Although not intending to limit the invention to any one method, cultures can be established from isolated low CD4 murine precursor cells using cell culture medium supplemented with a cytokine mixture said cytokines being TNFα, IL-lβ, IL-3, IL-7, SCF, FU3L, and CD40L. Said cultures exhibit initial cell death of approximately 25 to 30%. of the total cell number. After one day of culture, cell division and expansion commences at which stage cells have already begun to display cytoplasmic extensions and a general dendritic cell morphology. Cell expansion is maximal at 4 days of culture, representing 4 lo 5 times the original input, or around 7 times the initial surviving cell number. Al this time the cells are in the form of tight clusters. Greater than 95%» of the cells at this time have dendritic morphology. The number of viable cells declines after day 4 but a further live fold expansion can be achieved by splitting the cultures at day 3 to 4 and re-culluring in fresh medium with fresh cytokines.

Culluring murine pro-T cells under identical conditions similarly results in an extensive loss of cell viability over the first day amounting to approximately 70% of the initial cell input. After the initial cell drop, sub-division and expansion commence and cells of dendrilic morphology appear in the cultures. Cell expansion peaks al day 4 where cells are observed lo form light clusters and greater than 96% of said cells exhibit dendritic morphology. Dendritic cell expansion and development proceeds from the viable cells surviving following the first day of culture.

In a particular preferred embodiment said cytokine mixture comprises TNFα, IL-lβ, IL-3, IL-7, SCF and FU3L or functional derivatives thereof.

Another preferred cytokine mixture comprises TNFα, IL-lβ, IL-3, IL-7 and SCF or functional derivatives thereof.

Still another preferred cytokine mixture comprises TNFα, IL-l β, IL-3, IL-7, SCF, FU3L and CD40L or functional derivatives thereof. Yet another most preferred mixture comprises TNFα, IL-lβ, IL-3, IL-7, SCF, FU3L and anli-CD4() antibody or functional derivative thereof.

A most preferred mixture comprises TNFα, IL- l β, IL-3, IL-7, SCF, FU3L and FGK45.5 or functional derivatives thereof.

The process of the present invention may be homologous or heterologous with respect lo ihe animal or avian species from which the precursor cells and cylokines are derived. A homologous process means that Ihe species from which the precursor cells are derived is the same as the species from which the cylokines are derived. For example, murine cytokines used lo induce dendrilic cell development from murine precursor cells. A heterologous process is one where al least one cytokine in the mixture of cytokines employed is from a species different to the species from which the precursor cells are derived. For example, one or more human cytokines used lo induce dendritic cell formation from murine precursor cells or vice versa.

The present invention further contemplates a mixture for use in developing dendrilic cells from precursor cells said mixture comprising al lcasl three cytokines or functional derivatives thereof wherein at least two cylokines arc TNFα and IL-1.

More particularly the present invention contemplates a mixture for use in developing dendritic cells from precursor cells said mixture comprising at least three cytokines or functional derivatives thereof wherein at least three cylokines arc TNFα, IL- 1 and IL-3.

Most particularly said IL-1 is IL-lβ.

Preferably said mixture comprises the cytokine mixtures as hereinbefore described.

Even more preferably said precursor cells arc low CD4 precursor cells or pro-T cells. The present invention further extends to using the mixtures of cytokines lo induce dendritic cell development in vivo. Such a method may be particularly important for immune compromised individuals, subjects undergoing chemotherapy or radiation therapy, subjects undergoing transplantation procedures or subjects having cancer or a disease condition.

Accordingly, another aspect of the present invention contemplates a method of treating a subject said method comprising administering to said subject an effective amount of a mixture of at least three cytokines or functional derivatives thereof for a time and under conditions wherein said cytokines induce dendritic cell development from precursor cells.

Prelerably at least two cytokines are TNFα and IL-1.

Even more preferably at least three cytokines arc TNFα, IL- 1 and IL-3.

Most preferably said IL-1 is IL- l β.

In yet another aspect the present invention contemplates the use of a mixture of at least three cytokines in the manufacture of a medicament for the induction of dendrilic cell development from precursor cells in a subject wherein at least two cylokines are TNFα and IL-1.

More particularly the present invention contemplates the use of a mixture of at least three cytokines in the manufacture of a medicament for the induction of dendritic cell development from precursor cells in a subject wherein at lcasl three cylokines are TNFα, IL-1 and IL-3.

Most particularly said IL- 1 is IL-lβ. Anothcr aspect of the present invention contemplates a pharmaceutical composition comprising a mixture of at least three cytokines or functional derivatives thereof wherein said cytokines induce dendrilic cell development from precursor cells together with one or more pharmaceutically acceptable carriers and/or diluents.

Preferably at least two cytokines are TNFα and IL-1 .

Even more preferably at least three cytokines are TNFα, IL-1 and IL-3.

Most preferably said IL-1 is IL- l β.

Said cytokines or functional derivatives thereof are referred lo as ihe active ingredients.

The pharmaceutical forms suitable lor injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against Ihe contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, waler, ethanol, polyol (lor example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, lor example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, il will be preferable to include isotonic agenls, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent wilh various of the other ingredients enumerated above, as required, followed by appropriate action lo reduce microbial contamination, for example, the formulation may be filtered. Alternatively, a formulation may be prepared using sterilised components. In the case ol sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.

When the active ingredients arc suitably protected they may be orally administered, for example, wilh an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or il may be compressed inlo tablets. For oral therapeutic administralion, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least \ % by wcighl of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapculically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so thai an oral dosage unil form contains between about 0.1 μg and 2000 mg of active compound. Alternative dosages include from about 1 μg to about 100 mg and from about 10 μg lo about 500 mg.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereattcr: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium slearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of winlergreen, or cherry flavouring. When Ihe dosage unit form is a capsule, it may contain, in addition lo materials of the above type, a liquid carrier. Various other materials may be present as coalings or lo otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated wilh shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, melhyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations. The effective amount is that amount of individual or combined cylokines effective lo induce dendrilic cell development from haemopoietic cells. Proposed effective amounts include but are not limited to from about 0.01 /tg/kg body weight to about 1000 mg/kg body weight, or from about 700 mg/kg body weight or from 1 g/kg body weight lo about 500 mg/kg body weight. These amounts may be combined cytokine amounts or the amounts of individual cylokines used.

The pharmaceutical composition may also comprise genclic molecules such as a vector capable of transfecling target cells where the vector carries a nucleic acid molecule capable of modulating the expression of a nucleotide sequence encoding one or more cylokines or functional equivalent thereof. The vector may, for example, be a viral vector.

The present invention is exemplified herein with respect to murine precursor cells and murine dendritic cells. However, this is done with the understanding that the present invention extends to all animals including humans, livestock animals (e.g. sheep, cows, horses, donkeys), laboratory lest animals (e.g. rats, guinea pigs, rabbits, hamsters), companion animals (e.g. dogs, cats), captive wild animals (e.g. emus, kangaroos, deer, foxes) and birds (e.g. chickens, ducks, bantams, pheasants, emus, ostriches).

In yet another aspect the present invention contemplates the use of dendritic cells in the manufacture of a medicament for the treatment of a mammal.

The methods and compositions of the presenl invention are useful for generating dendritic cells for use in a range of therapeutic and diagnostic procedures. For example, the dendritic cells may be used as cellular vectors for anti-tumour and infectious disease vaccines or as induccrs of transplantation tolerance. These strategies arc based on the highly developed antigen presenting capacity of dendritic cells. The dendrilic cells of the present invention may also act as adjuvants for enhancing an immune response to, for example, tumour cells or other antigens such as those derived from prokaryoles, cukaryoles, or viruses, including human im uno deficiency viruses (e.g. HIV-I), influenza viruses and hepatitis viruses (e.g. Hepatitis A, B and C). In this regard, the dendrilic cells may also be loaded wilh such antigens lo induce tolerance. The dendritic cells of the present invention may also be used as regulators of the immune response for example by down-regulating T cell activity or skewing Ihe nature of a T cell response. Said down-regulation has applicability in the therapeutic or prophylactic treatment of disease conditions involving an unwanted immune response such as autoimmune conditions. Said skewing includes, for example, skewing a Thl dominated immune response lo a Th2 dominated immune response.

The dendritic cells produced by the method of Ihe present invention may be fused with other cell types such as, lor example, a tumour cell. Said fused dendritic cell is useful in a range of therapeutic and prophylactic procedures. For example, the processing and presentation of tumour antigens by a dendritic cell fused with a tumour cell has application as a therapeutic or prophylactic vaccine.

According to this aspect of the present invention said use of dendritic cells may be "syngeneic", "allogeneic" or "xenogcncic" wilh respect lo the individual undergoing treatment and the individual from which said precursor cells are initially derived. A syngeneic process means that the individual from which the precursor cells arc derived has the same MHC genotype as the individual receiving treatment. An allogeneic process means the precursor cells are derived from an individual which is MHC incompatible in relation lo the individual receiving treatment. For example, said precursor cells are derived from a Balb/C mouse while the individual receiving treatment is a CBA mouse. A xenogencic process means that the individual from which the precursor cells arc derived is ol a different species to the individual receiving treatment. For example, a primate is receiving treatment utilising dendritic cells derived from murine precursor cells.

Another aspect of the present invention contemplates a method of treating a subject said method comprising administering to said subject an effective number of dendritic cells wherein said dendritic cells are produced by the method of the present invention.

A further aspect ol^" the present invention contemplates dendritic cells produced by Ihe method of the present invention.

Further features of the present invention are more fully described in the following Examples and Figures. It is to be understood, however, lhat this detailed description is included solely for the purposes of exemplifying Ihe present invention. Il should not be understood in any way as a restriction on the broad description of the invention as set out above.

The present invention is further described by the following non-limiling Figures and Examples.

In the Figures:

Figure 1 is a graphical representation showing the stimulation of CD4 T cell proliferation by the dendrilic cell derived in culture from the thymic low

CD4 precursors. The cultured dendrilic cells were harvested on day 4 from cultures of Ihymic low CD4 precursors grown in the presence of Ihe cytokines IL-l β, TNFα, IL-3, IL-7, SCF, FH3L and Ihe mAb FGK45.5 reactive with CD40. These were compared with normal Ihymic dendritic cells extracted directly from the thymus, and finally purificd by sorting based on CDJ lc expression. Purified CBA lymph node CD4 T cells (20,000) were cultured for 3 days wilh various levels of the C57BL/6 derived dendrilic cells, then the cultures pulsed for 9 hr with ^H-TdR. The cells were collected onto glass- fibre filters and proliferation was assessed by measuring incorporated radioactivity using gas-flow scintillation counting. Results are the means ± SEM of the pooled data from two experiments, each with 5 cultures per point. Similar results but with somewhat lower counts were obtained at day 2.5 and 3.5 of harvest. The background count with T cells alone was 17 ± 1 cpm, the stimulation index was over 300. The background count wilh 2000 fresh thymic dendritic cells alone was 77 ± 22 cpm and with 2000 cultured dendritic cells alone was 109 ± 14 cpm.

Figure 2 is a graphical representation of the expression of CD8α on the dendrilic cells produced in spleen by transfer of various precursor populations.

Enriched splenic dendrilic cell preparations were stained for CD8α, class II MHC and Ly 5.2 expression; the histograms represent the level of CD8α on the dendritic cells, galed as high class II MHC cells wilh characteristic dendritic cell light scatter, and gated as Ly 5.2⁺ cells of donor origin. The broken lines gives ihe background fluorescence omitting only anti-CD8α. The upper graph demonstrates the presence of both CD8α and CD8α⁺ dendrilic cells in the spleens of normal mice. The second graph shows that the dendritic cell progeny of transferred bone marrow (BM) have a similar distribution of Cd8α and CD8α⁺ dendritic cell. The lower graphs demonstrate that the early thymic precursors generate only CD8α⁺ dendritic cells in the spleen following intrvenous transfer. This illustrates the selective production of one type of dendritic cell from the thymic precursors. EXAMPLE 1 Mice

The mice used for isolation of Ihymic low CD4 precursors, or for isolation of Ihymic dendritic cells, were usually 5-7 wk-old C57BL/6J Wehi females, bred under specific pathogen-free conditions at The Waller and Eliza Hall Institute animal facility. The GM-CSF null mice, produced at the Ludwig Institute , Melbourne (Stanley et al. , 1994), were originally on a C57BL/6 x 129 background but had been backcrosscd for 5 generations onto C57BL/6J mice; 5-9 wk old males and females were used. The source of the CD4⁺ lymph node T cells for mixed leukocyte reactions was 5-6 wk female CBA/J mice bred under specific pathogen-free conditions at The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Female C57BL/6J Wehi mice, 5-7 wks of age, bred under specific-palhogen-free conditions al The Walter and Eliza Hall Institute Animal Facility, were also used for the culture studies. In the cell transfer studies the precursor cells were isolated from C57BL/6 (Ly 5.2) mice and transferred into irradiated C57BL/6 Ly 5. rPep^1b mice as described fully elsewhere (Wu el al. , 1996; Wu et al. , 1991).

EXAMPLE 2

Isolation of Normal Dendritic Cells from the Thymus

The procedure was modified from that given in detail elsewhere (Vremac et al. , 1992; Suss & Shorlman, 1996; Winkel el al. , 1994). Briefly, pooled thymuses from 10 mice were cut into fragments and the entire tissue digested for 25 min at 22°C with collagenase DNasc. The digest was incubated a further 5 min wilh EDTA to break up dendrilic T cell complexes. Light density cells were then isolated from the digest by centrifugation at 4°C in a l .()77g/cm¹ density medium isoosmotic wilh mouse serum. The light density cells were then coaled wilh a cocktail ol mAb reactive with CD3, CD4, Thy 1 , CD25, B cell antigen B220, erylhrocyle antigen TER119, granulocyte antigen Gr-1 , macrophage antigen F480, FcRII and CDl lb, then the coated cells removed using anti-immunoglobulin coated magnetic beads. Finally the dendrilic cells in the enriched preparation were stained and stored as cells excluding propidium iodide, wilh the high forward and side scatter of dendritic cells, and expressing relatively high levels of CD1 lc (Suss & Shorlman, 1996).

EXAMPLE 3 Isolation of precursor cells from the thymus

Low CD4 - 2 methods were utilised:

(a) An improved procedure was used, modified from original method (Wu el al. , 1991) and given in detail elsewhere (Ismaili et al. , 1996). Briefly, the lightest 30%) of thymoeytcs were selected by a density cut procedure, then all adherent cells removed by culture for 1 hr in Pctrie dishes. Cells bearing CD3, CD8, CD25, B220, class II MHC, Gr-1 , Mac-1 and the crythrocyte anligen TER 119 were all removed by first coating wilh mAb, then removing coaled cells with two rounds of anti-immunoglobulin coaled magnetic beads. Finally, the 1% remaining Ihymocytes were stained with anli-Thy 1 (PE-conjugatcd 30. H12) and anti c-kit (FITC-conjugatcd ACK-2). The low CD4 precursors were sorted as cells low but positive for Thy 1 and moderate lo strongly positive for c-kit. The preparation was > 97% pure on reanalysis by these markers and appeared homogenous by 14 other markers tested in previous experiments (Wu et al. , 1991 ; Ardavin et al. , 1993; Wu el al. , 1995; Ismaili et al. , 1996; Shortman & Wu, 1996).

(b) The procedures have been described in full elsewhere (Wu el al. , 1991 ). The low CD4 precursors were isolated by first removing from thymus suspensions the 70%' most dense cells, then adherent cells, and then by immunomagnetic bead depletion removing mature T cells, CD4⁺8⁺ ihymocytes, most of the downstream precursors, and mature non-T lineage cells including dendritic cells. Anli-CD4 was nol employed in the depletion mAb cocktail. These precursors were then fluorescent labelled and finally positively sorted as cells which were low for Thy 1 and medium/high for c-kit.

Other Thymic precursors:

(c) The other precursors were isolated by first depleting thymus suspensions for cells bearing CD4, CD8 or CD3, as well as for mature non-T lineage cells including dendrilic cells. The precursors were then fluorescent labelled and positively sorted as cells which were c-kil CD25 ⁺ , or which were c-kit CD25⁺ or which were c-kit CD25 .

EXAMPLE 4 Culture Conditions

The culture medium was based on RPMI 1640, modified lo be isoosmolic wilh mouse serum (308m .osmolar), with additional Hepcs-buffcring at pH7.2, and supplemented with 10% v/v FCS, 10^'4M 2-ME, sodium pyruvate and antibiotics. The required cylokines were then added to the medium and the precursor cells dispersed in the mix. Cultures were from 1-7 days al 37.5"C in a humidified 10% v/v CO₂-in-air incubalor. For most studies the culture volume was 0.01ml, and culture of 1-3000 cells was in ihe wells of Tera.saki trays (Nunclon, Denmark). When a larger cell yield was required for cell counts or for surface phcnolype analysis, 20,000 precursors were cultured in 0.1 ml medium in flat-boltomed 96-wcll culture trays (Disposable Products Pty Ltd, South Australia).

EXAMPLE 5 Cytokines and Cytokine-related Antibodies

The following concentrations of recombinanl cylokines tested were used for culture of the low CD4 precursors: (a) IL-lβ (human), 200U or 0.2ng/ml; tumor necrosis factor (TNF)α (murine), lng/ml; IL-3 (murine), 200U or 400 ng/ml; IL-4 (murine), 200U or 20 ng/ml; IL-7 (human), 200U or lOng/ml; GM-CSF (murine), 200 "Immunex units" or 200ng/ml. GM-CSF was lound by lilration al the end of the experimental scries lo be 3200 "standard units" or the equivalent of 16 ng/ml of a standard Hall

Institute preparation.

(b) The medium was modi lied RPM I- 1640, isoosmolic wilh mouse serum, containing additional HEPES-buffcring al pH 7.2 and supplemented with 10% lclal calf serum, 10 ^""M 2-meιcaptoelhanol, sodium pyruvate and antibiotics.

The lull complement ol seven cytokines was used, namely: TNFα lng/ml; IL- lβ 0 2 ng/ml, IL-3 400 ng/ml, IL-7 100 ng/ml, SCF 10 ng/ml, Flt3 ligand 100 ng/ml, mAb against CD40, FGK45 5, 1 μg/ml. Cultures were incubated at 37.5°C in a humidilied 109? CO,-in-air gas phase.

These cylokines were all provided by Immunex Corp. , Seattle, WA, except for FH3/Flk2 ligand (FH3L), lOOng/ml, and antibody against GM-CSF, 2μg/ml, which were provided by Dr N Nicola, The Walter and Eliza Hall Institute of Medical Research Melbourne, Victoria, Australia, CD40 ligand (CD40L) and mAb against CD40, FGK45 5, 1 μg/ml, which were provided by Dr A Rolink, Basel Institute for Immunology, Basel, Switzerland

EXAMPLE 6 Cluster Counts, Cells Counts and Visualization of Dendritic Morphology

The incidence of dendritic cell clusters was counted diiectly on the Terasaki tray cultures, using inverted phase-contrast microscopy, a group of more than 20 cells was considered a cluster To recover and count cells after culture one tenth volume of 0 1M EDTA, pH 7 2, was first added to the warm cultures, then the cultures were mixed by repeated passage through a pipette tip in order to break up the dendritic cell clusters into a single cell suspension, cell counts were then carried out in a hemocytometer, using phase contrast microscopy To assess dendritic morphology, a cell suspension was prepared from pooled cultures using EDTA to aid dissociation, as above The cells were then washed by centrifugation through a layer of foetal calf serum, and resuspended in a small volume of culture medium The suspension was placed in slide chambers, prepared by fastening square coverslips onto microscope slides by double-sided adhesive tape at two opposite edges After filling the chambers the remaining edges were sealed with nail polish The slides were then incubated at 7°C for 1 -2 hr, then examined under phase- contrast microscopy To monitor the fate of individual precursor cells, Teraskai tray cultures containing only a single precursor were selected after 2 hr of incubation of cultures set up using 1 cell per 0 01 ml medium, then the culture was inspected every 24 hr using inverted phase-contrast microscopy

EXAMPLE 7 Iiiiimiiiofluoresceiit Staining and Flow Cytometry

The procedures used for staining the cultured dendritic cells were similar to those used previously for dendritic cells extracted from tissues (Vremac et al., 1992) The dendritic cells were harvested from the cultures al day 4 as above pooled, then stained with directly-conjugated mAb in two of three fluorescent colors, together with propidium iodide in order to exclude dead cells. They were then analyzed using a FACStar Plus (Becton Dickinson, San Jose, CA). The mAb and the fluorochromes used were. CD4, Cy 5 -conjugated H129. 19.6 8, CD8α, biotin-conjugated 53-6 7; CD8β, biotin conjugated 53-5 8, CD3, PE-conjugated KT3-1 1 , class I MHC, biotin-conjugated Ml/42, class II MHC, Texas Red-conjugated N22, CDl l c, biotin-conjugated N418, DEC205, FITC- conjugated NLDC145; BP-1 , biotin-conjugated 6C3, CDl lb, FITC-conjugated M 1/170; F480 macrophage antigen, biotin-conjugated F4/80, B220, biotin-conjugated RA3-6B2, CD80 (B7/1), biotin-conjugated I 6- 10A1 , CD86(B7/2), biotin-conjugated GL-1 , CD40, FITC-conjugated FGK45.5, CD44, FITC-conjugated IM7.81 , Gr-1, FITC-conjugated RB6-8C5, CD25, biotin-conjugated PC61 PE-conjugated steptavidin was used as the second stage for all biotin conjugates (Wu et al , 1991 , Wu et al., 1995, Vremec et al , 1992) EXAMPLE 8 Mixed Leukocyte Cultures for Assessing CD4 T cell Stimulatory Capacity

The cultures were set up and T cell proliferation determined as described previously (Suss 5 & Shortman, 1996). Briefly, 100-2000 dendritic cells of C57BL/6 origin, either harvested from the cultures or isolated from the thymus, were cultured with 20,000 purified CD4 T cells isolated from the lymph nodes of either CBA, C3H or C3H lpr mice The culture medium was modified RPMI- 1640, 0 1 ml being used in the wells of V-bottoni 96-well culture trays No exogenous cytokines were added After 2-4 days at 37 5°C in a 10 10%v/v-CO₂-in-air incubator, the cultures were pulsed for 9 hr with 'H-TdR Cells in the cultures were harvested onto glass-fibre filters and incorporated radioactivity measured in a gas-flow scintillation counter

EXAMPLE 9 15 Culture of Low CD4 Precursors with One or Two Cytokines

In the inventors' initial studies, the low CD4 precursors were isolated from adult mouse thymus by depletion then sorting, and then cultured at 50-1000 cells per well in Terasaki tray cultures with a range of recombinant cytokines The cytokines were tested singly, or 0 in combinations of two or sometimes three In no case of cytokines used alone or in pairs was any growth detected. This included the cytokines normally used to produce dendritic cells in culture, namely GM-CSF, or GM-CSF in combination with TNFα and/or IL-4. It also included IL-2 and IL-6, not used in subsequent studies

5 However, almost all the cytokines, even when used singly, gave some improvement in low CD4 precursor survival After 36 hr of culture in medium alone, an average of only 10% of the precursors were viable The cytokines which when used alone increased survival to above 30% where IL-3, IL-6, SCF, TNFα, IL-4 and GM-CSF; IL-7 gave the best survival, 55% No combination of cytokines gave a survival above 45%. Some 0 combinations of cytokines reduced survival to that of the medium alone, in particular TNFα with GM-CSF, TNFα with SCF NFα with IL-4, and IL-4 with IL-7 This indicated that low CD4 precursors expressed receptors for many of these cytokines but that the interactions between them were complex.

EXAMPLE 10 Growth and Differentiation of Low CD4 Precursors in

Response to Multiple Cytokines

In contrast to this lack of proliferation in response to combinations of up to two cytokines, some growth and differentiation of the low CD4 precursors was obtained on thymic epithelial cell lines (Saunders et al, 1995) A complex cocktail of seven cytokines, including some that might have been produced by thymic epithelial cells, namely TNFα, IL- l β, IL-3, IL-4, IL-7, SCF and GM-CSF was tested This cytokine cocktail was tested on the purified precursors cultured alone, without the thymic epithelial cell underlay. Purified thymic low CD4 precursors were cultured l r 4 days in 0.01 ml medium in Tera.saki tray cultures. The number of clusters ( > 20 cells) per well was counted under phase contrast microscopy. It produced a definite growth of precursors, doubling the input cell number by day 3. A requirement for multiple cytokines to induce growth in the low CD4 precursors has also been reported by Moore and Zlotnik (1995) However, the end-product cells appeared to be dendritic cells in the cultures of the present invention Cells with cytoplasmic extensions and dendritic cell morphology appeared by day 1 From day 2 to day 4 of culture a high proportion of the cells formed large clusters of around 50 cells, resembling closely the dendritic cell clusters generated by culturing bone-marrow or blood precursors with GM-CSF and other cytokines (Reid et al , 1990; Inaba et al., 1992; Caux e/ /., 1992; Scheicher e 1 al , 1992; Reid et al , 1992). The majority of cells in the cultures had the morphological appearance of dendritic cells by day 4

The clusters appeared to form as a result of aggregation, rather than representing true colonies derived from single precursors Nevertheless, over the range of 100-5000 low CD4 precursor cell input, there was a linear dose-response relationship between cells cultured and clusters formed at day 4, with 5-8 clusters being formed per 1000 cells cultured Accordingly, scoring the number of large clusters formed in the Terasaki well cultures provided a rapid assay for pi liferation and dendritic cell production With relatively dense cultures (3000 precursors per well) a statistically reliable estimate could be made with around 5 cultures pel point Such a cluster count was used as the initial readout for screening the contribution of different cytokines

EXAMPLE 11 The Effect of Omitting Cytokines on Dendritic Cell Cluster Development

To determine which of the cytokines in the complex mix were essential, the effect of leaving out one or two individual cytokines from the initial mix was systematically assessed, using the incidence of dendritic cell clusters at day 4 as a readout Thymic low CD4 precursor cells were cultured al 3000 cells per well in Terasaki culture plates, and Ihe number of clusters of dendrilic cells counted at day 4. Several cytokines (GM-CSF, IL4, IL-7, SCF and IL-3) could be omitted individually without much effect on cluster formation However, their absence generally did have an effect if omitted along with another cytokine, one exception was the omission of GM-CSF and IL-4 together, where no drop was evident The two cytokines whose omission, either alone or in combination with other cytokines, had the greatest effect were IL- l β and TNFα When omitted together, cluster formation dropped to 8% of that seen with the complete mix It was also notable that if GM-CSF was omitted, cluster formation became very dependent on IL-7 In contrast, the omission of both GM-CSF and IL-3, which share a common receptor chain (Nicola, 1994) and might therefore have substituted for one another, caused only a small drop in cluster formation

Accordingly IL-l β and TNFα were considered essential components of the mix, whereas GM-CSF and possibly IL-4 appeared dispensable To check this further GM-CSF was omitted from the mix and the effects of omitting one or two further cytokines (except L-l β and TNFα) was examined The further omission of IL-4 had no effect on cluster formation IL4 was omitted from subsequent cytokine cocktails The omission of IL-7 in the absence of GM-CSF now had a marked effect on cluster formation, although interestingly this drop was less when other cytokines, in particular IL-4, were absent EXAMPLE 12 Effect of Antibody Against GM-CSF on Development of Dendritic Cell Clusters

The lack of any requirement for GM-CSF in this production of dendritic cells was surprising, in view of its equii einent for dendrilic cell outgrowth in other systems It was possible the low CD4 precursors themselves, or some trace contaminant, produced sufficient endogenous GM-CSF To test this possibility, a neutralizing antibody against GM-CSF was added to the cultures when they were initiated, at a level known to block GM-SF-dependent colony-formation in culture The cultures were stimulated by the above "optimal" mix of five cytokines, lacking GM-CSF However, there was no significant drop in the number of dendritic cell clusters formed, nor any reduction in the apparent size of the clusters, when the antibody was added (Table 1 ) In view of the possibility that IL-3 was substituting for GM-CSF, since they share a common receptor chain (Nicola, 1994), the test was repeated with both IL-3 and GM-CSF omitted from the cytokine mix Again the anti-GM-CSF had no significant effect in these relatively high density cultures.

EXAMPLE 13 Cytokine Requirements in Low Cell Density Cultures

Finally, to verify the requirement for all five cytokines, some simpler combinations were tested, at a lower precursor cell input (250 precursors per culture) to reduce any effects of endogenous growth factor production The precursors were cultured in 0.01 ml medium in Tera.saki tray wells. Cell counts were performed after harvest in a hemocytomeler wilh viability assessed by appearance under phase-contrast microscopy. All simpler cytokine combinations gave fewer dendritic cell clusters, or no clusters at all (Table 2), and the few clusters that were obtained appeared smaller Two aspects of these low cell density cultures were notable First, neither dendritic cell cluster formation nor cell expansion was evident in cultures with IL- l β alone oi TNFα alone, or IL- l β plus TNFα, even though these cytokines were essential for the growth of dendritic cells in the cytokine mix However, in the cultures with IL- l β alone, or in cultures with IL- 1 β plus TNFα, approximately 20% of the individual, non-clustered surviving cells acquired dendritic morphology, suggesting IL- l β alone promoted some direct dendritic cell differentiation without cell division In accordance with this, a few dendritic cell clusters were obtained when very high density cultures were incubated in IL- l β plus TNFα A second aspect of the sparse cultures was the degree of dependence on IL-3 for dendritic cell cluster formation In contrast to the dense cultures where omission of IL-3 had a smaller and variable effect (Table 1 ), omission of IL-3 from the sparse cultures caused a much greater drop in both dendritic cell cluster formation and in cell proliferation A low level of endogenous IL-3 production by the precursor ceils themselves is one possible explanation for this difference

Under these low cell density culture conditions with the five cytokine mix, a net increase of the cells in the cultures was obtained, with growth extending to 5 days and reaching 3-4 times the original cell input Over 90% of the cells harvested at days 3 to 4 of culture had dendritic morphology In contrast, cultures of cells in medium without cytokines showed continuous cell death, no cell division and no cells with dendritic morphology

EXAMPLE 14 The Effect of CD40 Ligation and Flt3 Ligand Addition on Dendritic Cell

Development

Soluble CD40L has been found to enhance dendritic cell survival and differentiation (Caux el al., 1994) Although it was ineffective alone, the present inventors found that it enhanced the dendritic cell development stimulated by the above five cytokine mix In the presence of soluble CD40L, the cultured cells more rapidly attained the extreme dendritic cell form with extended dendrites, the number of clusters and their size was increased, the cell yield increased and fewer cells were found outside the clusters. The impression was of enhanced differentiation with an earlier peak of dendritic cell production Very similar results, but more reproducible in the extent of the effect, were obtained by adding the mAb FGK45 5, reactive with CD40 Cultures of 250 purified low CD4 precursors were set up in 0.01 ml medium in Terasaki tray culture wells. This mAb was used instead of soluble CD40L in subsequent experiments Flt3L injected into mice induces a striking increase in the levels of all types of dendritic cells in mouse lymphoid organs Although Flt3L was without effect on the low CD4 precursors alone, when added together with the previous five cytokine mix it enhanced dendritic cell development in culture The number of cells produced in the cultures increased 1 6-fold and peaked a day earlier, while the clusters increased a little in both number and size. The progeny cells again had dendritic cell morphology, although of a less extreme form than with CD40L.

The addition of both Flt3L and the mAb ligating CD40 to the cytokine mix of TNFα, IL- 1 β, IL-3, IL-7 and SCF, appeared to produce the optimal yield and morphological form of dendritic cells from the cultured low CD4 precursors, although these two additional "cytokines" were ineffective if used alone With this new seven "cytokine" mix the numbers of cells reached 4-5 fold the initial input by day 4 of culture, this was a minimal estimate, since under these conditions the clusters were difficult to completely dissociate even with EDTA. The number and size of clusters was also maximised, and peaked at day 4, with this mix Over 95% of the individual cells harvested and recovered from such cultures had the morphological appearance of dendritic cells, all having multiple fine cytoplasmic extensions and many having more obvious "dendrites".

EXAMPLE 15

CD4 Precursors from GM-CSF Null Mice

The surprising lack of any requirement for GM-CSF in dendritic cell generation required a more critical assessment It was possible that traces of endogenously derived GM-CSF persisted in the cultures despite the antibody blocking experiments of Table 1. It was also possible that GM-CSF was required for the generation of the precursors from multipotent stem cells, rather than at the later dendritic cell developmental steps reflected in our cultures. Accordingly, the development in culture of the thymic low CD4 precursors derived from GM-CSF "null" mice was assessed, with the GM-CSF gene deleted by homologous recombination (Stanley et al , 1994) The yield of low CD4 precursors from the thymus of the GM-CSF "null" mice was similar to that obtained from the normal C57BL/6 control mice, indicating that the generation of this precursor population was independent of GM-CSF When cultuied at low cell density with the final complement of seven "cytokines" (lacking GM-CSF), the thymic precursors from the GM-CSF null mice showed extensive proliferation and produced dendritic cell clusters, with over 90% of the product cells showing typical dendritic morphology (Table 3) However, both the total cell expansion and the number of dendritic cell clusters was only 70-75% that obtained by culturing the same number of low CD4 precursors from normal mice Thus, although dendritic cell development occurred efficiently in the absence of GM-CSF, it remained possible that traces of endogenous GM-CSF could enhance this development Alternatively, GM-CSF in the normal mice may have slightly "conditioned" the precursors for an enhanced response, or le oved some irrelevant cells from the "low CD4 precursor" pool

EXAMPLE 16 Effects of IL-3 and GM-CSF on Dendritic Cell Development in Low Cell Density

Cultures

The results with the GM-CSF null mice suggested that GM-CSF might have some stimulatory effect in low density cultuies, despite its lack of effect in the earlier high precursor cell input studies Another possibility was that the requirement for GM-CSF was being largely met by the added IL-3, via interaction with a common receptor β-chain, since the requirement for IL-3 only became pronounced in low density cultures (Table 2), it was important to recheck this issue under these conditions Accordingly, low CD4 precursors weie cultured with IL-l β, TNFα, IL-7, SCF, Flt3L and anti-CD40 mAb, then the effects of adding IL-3 and/or GM-CSF were examined (Table 4)

Precursor cell expansion and dendritic cell development occurred in the absence of both IL-3 and GM-CSF, with the vast majority of cultured cells having dendritic morphology and aggregating into clusters However, the yield of both dendritic cell and dendritic cell clusters was about half that seen in the presence of IL-3 Therefore, GM-CSF could partially substitute for IL-3 undei these conditions However, GM-CSF did not synergize with IL-3, as some inhibition in cell expansion was noted when both were added together Similar but slightly reduced effects were obtained when GM-CSF was added to the cultures at a 10-fold lower concentration

EXAMPLE 17

The Surface Phenotype of the Cultured Dendritic Cell

Immunofluorescent staining and flow cytoinetry was used to analyze the surface antigens on the cells obtained from day 4 cultures of low CD4 precursors grown in the mix of TNFα, IL-lα, IL-3, IL-7 and SCF, in the mix of these five cytokines together with Flt3L and the anti-CD40 mAb. The precursors (20,000) were grown for 4 days in 0.1 ml medium in 96-well, flal-botlomcd culture trays. The cultured cells were harvested, pooled, dissociated by EDTA treatment and then slained with mAb, generally in two fluorescent colours. Dead or damaged cells, approximately 15%* of the total, were excluded from analysis by propidium iodide staining and forward light scatter characteristics. The surface phenotype of the dendritic cells produced was similar under these two conditions. It was also largely unchanged when day 6 cultures were compared to day 4 cultures.

Of the characteristic T cell markers, the cells from these cultures lacked CD4, CD8α, CD8β, CD3, and CD25. This implied they had lost the low level of CD4 characteristic of the precursors, but had not progressed to the next downstream CD25+ precursor, nor had they formed mature T cells Surprisingly, they had not gained CD8α, a marker characteristic of most thymic dendritic cells The cells were Thy 1 positive, showing a wide range of Thy 1 expression; however, since many types of cultured cells express Thy 1, this marker is not useful for defining T lineage cells in culture

Of the B lymphocyte lineage markers, the cells lacked B220, and also lacked BP- 1, an early B-cell marker expressed on thymic dendritic cells However, BP-1 is known to be induced on dendritic cells by the thymic environment, and the low CD4 precursors produce BP-1 -DC if allowed to develop in the spleen Of the macrophage/granulocyte markers, the cells were negative for Gr-1 They stained at levels varying from negative to moderately positive for CDl lb (Mac- 1 ) and negative to moderately positive for F4/80 The veiy strong staining characteristic of macrophages was not seen with either marker CDl l b is expressed at levels ranging from low to high on different lymphoid tissue dendritic cells (Ismaili el al., 1996, Shortman & Wu, 1996) F4/80 has been observed on cultured dendritic cell precursors, but is not normally expressed at this level on mature tissue dendritic cell (Vremac et al , 1992)

Of the typical dendritic cell markers, the cultured cells expressed very high levels of class I and class II MHC, high levels of CD l l c and moderate levels of DEC205 They expressed B7- 1 (CD80) and B7-2 (CD86), characteristics of mature dendritic cells As do most dendritic cells, they expressed CD40, CD44 and HSA, the heat stable antigen, these markers are also found on other cell types They also had the high forward and side scatter characteristic of dendritic cells, as expected from their size and appearance in Overall they resembled mature dendritic cells, the only anomaly being the absence of CD8, a marker characteristic of thymic dendritic cells in the mouse but present on only about half of splenic or lymph node dendritic cells However, there is no evidence that CD8 has any role in either the function or the development of dendritic cells It should be noted that the low CD4 precursors lack surface class II MHC, CDIIc, DEC205, CD80 and CD86, so differentiation towards expression of these dendritic cells markers had occurred in the cultures

EXAMPLE 18 T Cell Stimulatory Activity of the Cultured dendritic cells

To determine if the culture system produced functional dendritic cells, the dendritic cells produced from thymic precursors by culture in the presence of IL-l β, TNFα, IL-3, IL-7, SCF, Flt3L and mAb binding CD40 were compared with freshly isolated thymic dendritic cells in their ability to stimulate the proliferation of allogeneic CD4 T cells The small scale mixed leukocyte cultures used (Suss & Shortman, 1996) involved 20,000 pure CD4⁺ lymph node T cells as responders, and small numbers of either pure thymic dendritic cells or cultured dendritic cells as stimulators, no exogenous cytokines were added Both the dendritic cells cultured with the full seven cytokine mix and the normal thymic dendritic cells were found to be efficient stimulators of matute T cell proliferation, both gave a proliferative response peaking at day 3 to 3 5, and both gave a good dendritic cells 5 dose/response relationship and a very high stimulation index (Fig 1 ) The dendritic cells cultured with the full cytokine mix gave a slightly bettei T cell stimulation than freshly isolated thymic dendritic cells Dendritic cells cultuied in the absence of Flt3L and anti-CD40, although giving good stimulation at low dendritic cells levels, gave a reduced proliferation compared to freshly isolated thymic dendritic cells above 1000 dendritic cells 10 per culture

EXAMPLE 19 Frequency of Responding and Dendritic Cells-Producing Precursors

15 The rapid increase in cell counts during the response of the low CD4 precursor preparation to the cytokine mixes suggested that a significant proportion of the cells were responding However, since it required around 50 precursors to form one dendritic cell cluster at day 4, it may have been that only 2% of the precursor population was committed to dendritic cell development The aigument against this was that the incidence of cells

20 with dendritic cell morphology was very much higher than 2% early in the culture, and that the clusters represented aggregates, rather than colonies derived from a single precursor To determine the actual incidence of responding cells, a series of experiments was conducted where the low CD4 precursors were set up in Terasaki tray cultures at the 1 cell per well level in medium containing the optimum mix of "cytokines" (IL- l β, TNFα,

25 IL-3, IL-7, SCF, Flt3L and anti-CD40 antibody) After 2 hr the cultures were examined under inverted phase microscopy, and cultures with a single precursor in the well were selected for further day by day observation In one such experiment the fate of 27 single precursor cells was followed over 3 days By day 1 , 9 of the starting cells had died, but all but one of the remaining viable cells showed a clear dendritic cell morphology, of these

30 70% survivors with dendritic cell morphology, 20% had already undergone one cell division By day 3, one further culture had terminated in cell death, but all the remainder had divided to produce between 2 and 10 progeny, 90% of the viable progeny cells in these clones were of dendritic cell morphology In some cases the progeny cells stayed associated as a mini-cluster, but in many cultures they moved apart. Two subsequent experiments confirmed these observations. Thus the majority (at least 70%) of the cells in 5 the low CD4 precursor preparation could be considered as potential dendritic cell precursors It was of interest that differentiation of these low CD4 precursors to a dendritic cell morphology was rapid and usually preceded cell division.

EXAMPLE 20 10 Growth and Dendritic Cell Development from Successive T Precursor Populations

When the earliest adult mouse thymus T precursor population, the low CD4 precursor (CD4'"8^'3^"25 44⁺c-kit⁺), was cultured in the seven cytokine mix, extensive cell growth and dendritic cell development occurred as described previously (Saundcrs et al. ,

15 1996). Some initial cell death occurred, shown previously by single cell culture to represent 25-30% of the total cells. After one day cell division and expansion commenced; al this stage cells had already began lo display cytoplasmic extensions and a general dendritic cell morphology. Cell expansion was maximal at day 4 of culture, representing 4 to 5-times the original input, or around 7-times the initial surviving cell 0 number. At this lime the cells were in the form of light clu.sters, difficult to completely dissociate, so these counts may have been an underestimate. Almost all ( > 95% ) of the cells at this time had dendritic morphology under phase contrast microscopy. The numbers of viable cells declined after day 4, but a further 5-fold expansion could be achieved by splitting the cultures al day 3-4 and reculluring in fresh medium with fresh 5 cylokines. However, after a second recullurc, dendritic morphology was lost, cell expansion was reduced and extensive cell death occurred.

When the next downstream T precursor population, ihe pre-T cells (CD4 83 25⁺44⁺c- kil⁺) (Shortman & Wu, 1996; Godfrey & ZIotnik, 1993), was cultured under identical 0 conditions, there was a more extensive loss of cell viability over the first day, amounting to around 70% of the initial cell input. Alter this initial cell drop, cell division and expansion commenced al day 1 and cells wilh dendrilic morphology appeared in the cultures. The cell expansion reached a peak at day 4, where the cells were in the form of tight clusters and > 969. of the cells had dendritic morphology. Although the overall yield of cells compared lo the total initial input was much lower than with the low CD4 precursors, there was a similar level of expansion and dendritic cells development from the viable cells surviving at day 1 . The main difference between the cultures of the two precursor populations was therefore the level of initial cell death.

When the third successive mouse thymus precursor population, the pre-T cell (CD4 8 3 25⁺44 c-kit^") (Shortman & Wu, 1996; Godfrey & Zlotnik, 1993), was cultured, very different results were obtained. The cells showed an extensive loss of viability over the first day and this loss continued thereafter. No cell expansion was obtained and there were few, if any, dividing cells in the cultures. Of the viable cells that survived, very few exhibited dendritic morphology.

When the final precursor population, the "late double negatives" (CD4 8 3 25 44 c-kit ) (Shorlman & Wu, 1996; Godfrey & Zlotnik, 1993), was cultured, the results differed slightly from thai obtained with the previous precursor population. Again there was extensive initial cell death. However, a proportion of the cells did proliferate and some expansion of cell numbers was evident by day 4. This expansion was more marked in more concentrated "bulk" cultures than in the 250 cell Terasaki cultures, suggesting il was in parl driven by endogenously produced cylokines. However, despite this limited cell expansion, very few of the cells displayed dendrilic morphology. EXAMPLE 21 Surface Markers on Cultured Cells Derived from Successive T Precursors

The cell surface phenotype o the cultured cells was determined by harvesting al day 4, immunofluorcscent staining and flow eytomclric analysis. The surface phenotype of the cells derived from the low CD4 precursors and the pro-T cells was similar, and confirmed that in both cases the cells produced were dendrilic cells. They expressed high levels of class II MHC, of CD l lc and of CD44. They were positive for DEC- 205, although the level of expression was lower than for normal thymic dendritic cells. They expressed moderate levels of CD80 (B7/1) and CD86 (B7/2), suggesting they were mature dendritic cells. They lacked T cell or B cell markers and were relatively low or negative for myeloid markers. CDl lb was expressed, but this is present at moderate levels on certain dendritic cells and is upregulated on all murine lymphoid- organ derived dendrilic cells on culture.

In contrast, the cells surviving at day 4 in cultures of the pre-T cells or the late double negatives were class II MHC low and CD l lc low, confirming the morphological assessment that they were not dendrilic cells.

The surface phenotype analysis gave some clues to the nature of the few cells produced by proliferation in the late double negative precursor cultures. They expressed low levels of CD4 and a proportion expressed both CD8α and CD8β, many expressed CD3 and a small proportion expressed TCRγδ. This suggested many of the proliferating cells were of the T lineage, and some were γδ T cells. In previous results from this laboratory γδ T cells have been produced in culture from late double negatives under the influence of IL-2 (Petrie et al. , 1992). Thus the limited expansion and differentiation obtained from this population may have been driven by endogenously produced IL-2, in conjunction with some of the cytokines added. However, the cytokine mix employed was clearly nol optimal for T-cell differentiation from these late precursors, given the limited proliferation and differentiation achieved. EXAMPLE 22 Yield of dendritic cells from Different T Precursor Populations: in vivo and in vitro

Comparison

The yield of dendritic cells from the successive T precursors around the peak of the response, either at 4 days of culture or in recipient spleens or thymuses 14 days alter intravenous transfer, is compared in Table 5. The cell culture and the cell transfer results agree in ihe general conclusion that dendritic cells can be produced from both the low CD4 precursors and the pro-T cells, but not from the pre-T cells or the late double negatives. There is some disagreement regarding the relative efficiency of dendritic cells production by the two earlier precursor populations, the culture results indicating a marked reduction as precursors develop lo the CD4^"8^'25⁺44⁺c-kit⁺ stage whereas the transfer studies suggest a near equivalent efficiency in the Ihymus but a substantial drop in efficiency in the spleen. This might be the result of the extensive death of the pro-T cells in culture, which may not occur in vivo. Alternatively, dif ferences in Ihe efficiency of seeding different organs by the two precursor populations could explain the discrepancy. The differences in the peak havesl time for dendrilic cell development (4 days in culture, 7- 14 days in the thymus or spleen) is a third variable.

EXAMPLE 23 The Expression of CD8α and BP-1 on Thymic dendritic cells Generated in vitro or in vivo

The lack of any expression of BP- 1 , and the very low lo negative expression of CD8α on the dendrilic cells generated in culture from the Ihymic precursors presented a paradox. These two surface proteins, and the mRNAs for their formation, arc expressed by normal thymic dendritic cells (Wu et al. , 1995; Vrcmac el al. , 1992). We have shown previously (Wu et al. , 1996) that BP-1 is expressed on Ihe dendrilic cells progency of the low CD4 precursors if they seed in the thymus, but nol if they sced in Ihe spleen, indicating that some factor in the the Ihymus induces expression of this marker; perhaps it is not surprising that this factor is missing in culture. However, CD8α is present on the dendrilic cells progency of both early precursors, regardless of whether they develop in the thymus (Wu el al. , 1995 ; 1996) or in the spleen (Fig. 2). In contrast, the dendritic cells which develop from these ihymic T precursors in culture resemble conventional myeloid-derived dendritic cells by most surface markers and lack CD8α In view of the high efficiency of dendrilic cell production in culture il seems unlikely that different subpopulations of the precursors function in vitro than function in vivo. It is more likely that a factor inducing CD8α expression is present in both spleen and thymus, but missing in our cultures. In support of this, the dendritic cells progeny found in the lymph nodes after intravenous transfer of these precursors includes a proportion of CD8α and CD8α'" dendritic cells, as well as the CD8α^hl dendritic cells. Attempts lo induce CD8α expression on the cultured dendrilic cells by the addition of other cytokines (including TGFβ) have so far proved unsuccessful. However, there is no evidence that CD8α is essential for either the development or the function of dendritic cells.

EXAMPLE 24 Dendritic cell production in culture: different precursors, different cytokines

Precursor cells, either the mouse Ihymic low CD4 precursor or mouse bone marrow cells depleted of all cells bearing lineage markers of mature blood cells (Lin^' Bone Marrow, an enriched source of precursor cells) (Wu el al. , 1991 ; Wu et al. , 1996) were cultured at a level of 250 cells per well in Terasaki culture trays. The cultures were stimulated either wilh GM-CSF and TNFα (Ihe cytokine mix usually used lo grow myeloid-related dendrilic cells) or wilh the mix of cylokines found to be optimum for production of dendritic cells from the Ihymic lymphoid-precursor populations [TNFα, IL-lβ, IL-3, IL-7, SCF, FU-3L, anli-CD40 antibody). At day 4, the total number of cells in the cultures showing dendritic morphology was counted under phase contrast microscopy . Results are means ± SEM of 20 cultures. GM-CSF and TNFα gave signi leant dendrilic cell production from bone marrow precursors, but not from the thymus precursors (Table 6). The seven cytokine mix lacking GM-CSF grew out dendritic cells I rom both sources. The results suggest the thymic precursors were of one type only (lymphoid-relalcd precursors) whereas the bone marrow precursors were of two types (lymphoid-related and mycloid-relaled precursors). The results also indicate that dendrilic cell precurors responsive to the seven cytokine mix are present in tissues other than the thymus.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described It is to be understood that the invention includes all such variations and modifications The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually oi collectively, and any and all combinations of any two or more of said steps or features

TABLE 1

The effects of antibody against GM-CSF on the generation of dendritic cells clusters by cultured thymic low CD4 precursors

Cytokine mix Anti-GM-CSF Clusters per well

20±2

TNFα- IL-1 β+IL-7+SCF+IL-3

+ 18±2

22±2

TNFα+IL-l β+IL-7+SCF

+ 19*1

Purified thymic low CD4 precursors were cultured at 3000 cells per well in 0.01 ml medium for 4 days with the cytokines liste Results are the means ± SEM of pooled data from 3 experiments, each with 5 cultures per assay.

TABLE 2

The effects of various cytokine combinations on the generation of dendritic cells clusters in low density cultures of thymic precursors

Cytokines Clusters per culture

No cytokines O±O lL-l β O±O

TFNα O±O

TNFα+IL-l β O±O

IL-l β+TNFα+SCF O±O

IL-l β+TNFα+IL-3 0 4±0 6

IL-l β+TNFα+IL-7 0 l±O 2

IL- 1 β+TNFα+IL-7+SCF 0 1 ±0 2

IL- 1 β+TNFα+IL-7+IL-3 0 2±0 4

IL- 1 β+TNFα+SCF+IL-3 0 4±0 6

1L-7+SCF+1L-3 0 l±O 1

IL-l β τ TNFα+IL-7+SCF+IL-3 2 O±O 9

Cultures of 250 low CD4 precursors were set up in 0 01 ml medium in Terasaki tray wells The incidence of dendritic cells clusters was determined after 4 days of culture Results aie the means ± SEM of data pooled from 2 experiments, each with 20 cultures per condition TABLE 3

The growth and development of dendritic cells clusters from thymic low CD4 precursors from GM-CSF "null" mice

Control C57BL/6 GM-CSF null precursors precursors

Cells per culture 1002± 109 721 ±69

Clusters per culture 5 7± l 6 4 7±1 6

Purified low CD4 precursors, 250 per 0 01 ml medium in Terasaki culture wells, were cultured for 4 days in the presence of TNFα, IL- l , IL-3, IL-7, SCF, Fit3L and FGK45 5 mAb against CD40 Results are the means ± SEM of pooled data from 2 experiments, each with 20 cultures per determination

TABLE 4

The influence of GM-CSF and IL-3 on dendritic cells development from low CD4 thymic precursors in low density cultures

Cytokines Cells per culture Clusters per day 4 culture day 4

IL- l β, TNFα, IL-7, SCF, FK-3L, anti-CD40 4,930 1 8±0 4 alone

IL- l β, TNFα, IL-7, SCF, FU-3L, anti-CD40 10, 130 4 3±0 4 plus IL-3

IL- l β, TNFα, IL-7, SCF, F1.-3L, anti-CD40 6,570 3 7±0 4 plus GM-CSF

IL-l β, TNFα, IL-7, SCF, FU-3L, anti-CD40 8,360 4.6±0.5 plus IL-3 and GM-CSF

Purified low CD4 thymic precursors were cultured at 250 cells per well in 0 01 ml medium for 4 days with the cytokines listed Results are the pooled data from 2 experiments, each with 20 cultures per condition Cluster counts are the means ± SEM of individual direct culture counts. Cell counts were performed on the pool of the 20 cultures in each experiment after harvest and the results are the mean of the 2 experiments TABLE 5

Precursor Culture Transfer i.v. population DC per 10" Thymus Spleen precursors DC per 10" precursors day 4 day 14

CD4^,n 6.1 ± 2.3 x 10" 0.4 ± 0.1 x 10" 0.4 ± 0.7 x 10"

CD25^' c-kit^"- 1.8 ± 0.8 x 10" 0.4 ± 0.2 x 10" 0.4 ± 0.1 x 10"

TABLE 6

Dendritic Cell Production

TNFα. ILL IL3, IL7 GM-CSF SCF. H13L. anli-CD40 + TNFα

Low CD4

Thymus precursor 906 ± 57 2 ± 3

Lin-Bone marrow 864 ± 37 641 ± 69

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Claims

CLAIMS:

1. A method of developing dendritic cells from precur.sor cells, aid method

comprising culturing said precur.sor cells in the presence of an effective amount of a

mixture of al least three cytokines or functional derivatives thereof wherein at lcasl Iwo

cytokines are TNFα and IL-J or functional derivatives thereof.

2. A method according to claim 1 wherein al lcasl three cytokines are TNFα, IL-1

and IL-3 or functional derivatives thereof.

3. A method according to claim I or 2 wherein said IL-1 is IL-lβ or a functional

derivative thereof.

4. A method according to claim 3 wherein said mixture comprises TNFα, IL-lβ

and IL-3 and an effective number of cylokines selected from IL-7, SCF, FU3L,

CD40L and an anti-CD4() antibody, or functional derivatives thereof.

5. A method according lo claim 4 wherein said mixture comprises TNFα, IL-lβ,

IL-3, IL-7, SCF and FH3L or functional derivatives thereof.

6. A method according to claim 4 wherein said mixture comprises TNFα, IL-l β,

IL-3, IL-7 and SCF or functional derivatives thereof.

7. A method according to claim 4 wherein aid mixture comprises TNFα, IL-lβ,

IL-3, IL-7, SCF, Flt3L and CD40L or functional derivatives thereof.

8. A method according lo claim 4 wherein said mixture comprises TNF-α, IL- lβ,

IL-3, IL-7, SCF, F1.3L and an anli-CD40 antibody or functional derivatives thereof.

9. A method according to claim 4 wherein said mixture comprises TNFα, IL-lβ,

IL-3, IL-7, SCF, FH3L and FGK45.5 or functional derivatives thereof.

10. A method according lo claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 wherein

said precursor cells are lymphoid precursor cells.

11. A method according to claim 10 wherein said lymphoid precursor cells are T

cell precursor cells.

12. A method according to claim 1 1 wherein said T cell precursor cells are CD4

low precursor cells.

13. A method according to claim 1 1 wherein said T cell precur.sor cells are pro-T cells.

14. A mixture for use in developing dendrilic cells from precursor cells said mixture comprising at lea.st three cylokines or functional derivatives thereof wherein at least two cylokines are TNFα and IL-1 .

15. The mixture according lo claim 14 wherein at least three cytokines are TNFα,

IL- 1 and IL-3 or functional derivatives thereof.

16. The mixture according to claim 14 or 15 wherein IL-1 is IL-l β or a functional

derivative thereof.

17. The mixture according to claim 16 wherein said mixture comprises TNFα, IL-

l β and IL-3 and an effective number of cytokines selected from IL-7, SCF, FH3L,

CD40L and an anli-CD40 antibody or functional derivatives thereof.

18. The mixture according to claim 17 wherein said mixture comprises TNFα, IL-

lβ, IL-3, IL-7, SCF and Flt3L or functional derivatives thereof.

19. The mixture according lo claim 17 wherein said mixture comprises TNFα, IL-

l β, IL-3, IL-7 and SCF or functional derivatives thereof.

20. The mixture according lo claim 17 wherein said mixture comprises TNFα, IL-

l β, IL-3, IL-7, SCF, FU3L and CD40L or functional derivatives thereof.

21 . The mixture according to claim 17 wherein said mixture comprises TNF-α, IL-

l β, IL-3, IL-7, SCF, F1.3L and an anti-CD40 antibody or functional derivatives thereof.

22. The mixture according to claim 18 wherein said mixture comprises TNFα, IL-

lβ, IL-3, IL-7, SCF, FU3L and FGK45.5 or functional derivatives thereof.

23. A method of treating a subject said method comprising administering lo said

subjet an effective amount of a mixture of at least three cylokines or funclional

derivatives thereof for a lime and under conditions wherein said cylokines induce

dendrilic cell development from precursor cells.

24. A method according lo claim 23 wherein al least two cytokines arc TNFα and

IL-1 or funclional derivatives thereof.

25. A method according lo claim 23 wherein al least three cytokines arc TNFα, IL-

1 and IL-3 or functional derivatives thereof.

26. A method according lo claims 24 or 25 wherein IL-1 is IL-lβ or a funclional

derivative thereof.

27. Use of a mixture of al least three cylokines in Ihe manufacture of a medicament

for the induction of dendritic cell development from precursor cells in a subject wherein

at lea.st two cytokines are TNFα and IL-1 or funclional derivatives thereof.

28. Use of a mixture ot at least three cytokines in the manufacture ol a medicament

for the induction of dendritic cell development f rom precursor cells in a subject wherein

at least three cylokines are TNFα, IL- 1 and IL-3 or functional derivatives thereof.

29. Use of a mixture according to claims 27 or 28 wherein IL-1 is IL-l β or a

functional derivative thereof.

30. A pharmaceutical composition comprising a mixture of al least three cylokines

or functional derivatives thereof wherein said cytokines induce dendritic cell

development from precursor cells together wilh one or more pharmaceutically

acceptable carriers and/or diluents.

31. Λ pharmaceutical composition according lo claim 30 wherein at least two

cytokines are TNFα and IL-1 or f unctional derivatives thereof.

32. A pharmaceutical composition according to claim 30 wherein at least three

cytokines arc TNFα, IL-1 and IL-3 or lunctional derivatives thereof.

33. A pharmaceutical composition according lo claims 31 or 32 wherein said IL-1 is

IL-lβ or a functional derivative thereof .

34. Use of dendritic cells in the manulaclure ol a medicament lor the treatment of a

subject.

35. A method of treating a subject said method comprising administering to said

subject an effective number of dendritic cells wherein said dendritic cells arc produced

by the method of the present invention.

36. The dendrilic cells produced by Ihe method of claim 1.

37. An agent for use in developing dendrilic cells from precursor cells comprising a

mixture of al least three cylokines or functional derivatives thereof wherein at least two

cytokines arc TNF-α and IL- 1 or functional derivatives thereof.

38. An agent according to claim 37 wherein said IL-1 is IL-lβ or a funclional derivative thereof.