WO2004015056A2 - Immunomodulating compositions, processes for their production and uses therefor - Google Patents
Immunomodulating compositions, processes for their production and uses therefor Download PDFInfo
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- WO2004015056A2 WO2004015056A2 PCT/AU2003/001021 AU0301021W WO2004015056A2 WO 2004015056 A2 WO2004015056 A2 WO 2004015056A2 AU 0301021 W AU0301021 W AU 0301021W WO 2004015056 A2 WO2004015056 A2 WO 2004015056A2
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- antigen
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Definitions
- THIS INVENTION relates generally to modulation of immune responses. More particularly, the present invention relates to compositions and methods for antigen-specific suppression of immune responses, including primed immune responses. Even more particularly, the invention is directed to the use of antigen-presenting cells, especially dendritic cells, whose level and or functional activity of CD40, or its equivalent, is impaired, abrogated or otherwise reduced, for treating and/or preventing unwanted or deleterious immune responses including those that manifest in autoimmune disease, allergy and transplant rejection.
- Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease.
- T cell interaction as a therapeutic approach to human autoimmune disease lags behind the blockade of pro-inflammatory and tissue destructive cytokines.
- Blockade of products of the innate immune system including TNF- ⁇ and IL-ip, produces dramatic anti-destructive clinical effects in the autoimmune disease rheumatoid arthritis (RA) (reviewed by (Feldmann and Maini, 2001)).
- RA rheumatoid arthritis
- Novel approaches include reinstitution of control on self-antigen presentation, derived from regulatory T cells (Treg). Suppression of immune effector cells by a variety of described Treg is a key mechanism for peripheral tolerance (Maloy and Powrie, 2001; Roncarolo and Levings, 2000).
- the complex interactions resulting in the generation of T cell-mediated immune responses are dependent on antigen presentation, cognate interactions between T cells and antigen presenting cells (APCs) including dendritic cells (DCs), and the concomitant production of soluble and membrane costimulatory molecules by APCs and T cells (reviewed by (Banchereau et al., 2000)).
- APCs antigen presenting cells
- DCs dendritic cells
- soluble and membrane costimulatory molecules by APCs and T cells
- Various drugs and cytokines, and inhibitors of NF-cB have been shown to inhibit myeloid DC maturation (de Jong et al, 1999; Griffin et al, 2001;hackstein et al, 2001; Lee et al, 1999; Mehling et al, 2000; Steinbrink et al, 1997; Yoshimura et al, 2001).
- DCs generated in the presence of these agents altered T cell function in vitro and in vivo, including promotion of allograft survival (Giannoukakis et al, 2000; Griffin et al, 2001). Despite this, suppression of previously primed CD4 + T cell responses by DCs in vivo has not been demonstrated. This is important for therapy of pre-existing autoimmune disease as CD4 + effector T cells mediate the perpetuation of tissue damage in autoimmune disease through their interaction with monocytes, B cells and local DCs (Feldmann, 2001; MacDonald et al, 1997; Sakata et ⁇ /., 1996).
- the present invention is predicated in part on the unexpected discovery that inhibition of NF-/cB activity, especially RelB inhibition, in precursors of APCs leads to the production of APCs with reduced or abrogated CD40 expression, which can not only prevent priming of immunity but which can also suppress a previously primed immune response.
- these APCs induce the differentiation of CD4 + regulatory T cells that can transfer tolerance to antigen-primed recipients in an IL-10 dependent manner.
- the foregoing discovery has been reduced to practice in the form of immunomodulating compositions and methods of treatment or prophylaxis, as described hereinafter.
- an isolated antigen-presenting cell for modulating an immune response which is characterised by producing CD40, or its equivalent, at a level and/or functional activity which is lower than that produced by an activated dendritic cell.
- the antigen-presenting cell is other than a B lymphocyte.
- the antigen-presenting cell produces CD40, or its equivalent, at a level and/or functional activity that is less than about 1% of that produced by an activated dendritic cell.
- the antigen-presenting cell produces CD40, or its equivalent, at a level and/or functional activity that is lower than that produced by an immature dendritic cell.
- the antigen-presenting cell is preferably selected from monocytes, macrophages, B lymphocytes and their precursors as well as other cells of myeloid lineage, dendritic cells or Langerhans cells but is more preferably selected from dendritic cells and macrophages.
- the antigen-presenting cell is further characterised by producing NF- ⁇ B or component thereof, especially RelB, at a level and/or functional activity which is lower than that produced by a mature or activated dendritic cell.
- the antigen-presenting cell produces NF- ⁇ B or component thereof, especially RelB, at a level and/or functional activity that is lower than that produced by an immature dendritic cell.
- the antigen-presenting cell is further characterised by producing an immunostimulatory molecule, especially CD86 or its equivalent.
- the immunostimulatory molecule is produced at a level and/or functional activity which is at least about 10% of, but preferably about the same as, that produced by an activated dendritic cell.
- the antigen-presenting cell is produced by a process comprising contacting a precursor of the antigen-presenting cell with an NF- ⁇ B inhibitor for a time and under conditions sufficient to differentiate an antigen-presenting cell from the precursor and to inhibit or otherwise reduce the level and/or functional activity of NF- ⁇ B in said cell.
- the precursor is preferably derived from monocytes or bone marrow.
- the NF- ⁇ B inhibitor inhibits nuclear translocation of NF- ⁇ B or component thereof, especially RelB.
- the antigen-presenting cell is produced by a process comprising contacting an antigen-presenting cell, or its precursor, with an inhibitor of CD40, or its equivalent, for a time and under conditions sufficient to produce a modified antigen-presenting cell that produces CD40, or its equivalent, at a reduced or abrogated level and/or functional activity relative to that of said antigen-presenting cell or its precursor.
- the process is further characterised by contacting the antigen-presenting cell, or its precursor, or the modified antigen-presenting cell, with an agent that increases the level and/or functional activity of an immunostimulatory molecule, especially of CD86 or its equivalent, for a time and under conditions sufficient to enhance or otherwise elevate the level and/or functional activity of the immunostimulatory molecule in the antigen-presenting cell, or its precursor, or the modified antigen-presenting cell.
- an agent that increases the level and/or functional activity of an immunostimulatory molecule especially of CD86 or its equivalent
- Another aspect of the present invention contemplates a method of producing antigen- presenting cells for modulating an immune response to a target antigen, comprising contacting an antigen-presenting cell as broadly described above with an antigen that corresponds to the target antigen, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen- presenting cell.
- the antigen presentation is restricted by major histocompatability (MHC) molecules.
- MHC major histocompatability
- the mvention encompasses an antigen-specific antigen-presenting cell for modulating an immune response to a target antigen, which is produced by contacting an antigen-presenting cell as broadly described above with an antigen that corresponds to the target antigen, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen- presenting cell.
- the invention provides a method of producing antigen-presenting cells for modulating an immune response to a target antigen, comprising contacting a precursor of the antigen-presenting cell with an NF- ⁇ B inhibitor and with an antigen that corresponds to the target antigen, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient to differentiate an antigen-presenting cell from the precursor and to inhibit or otherwise reduce the level and/or functional activity of NF- ⁇ B in said cell, wherein the antigen or a processed form thereof is presented by the antigen-presenting cell so produced.
- the antigen-specific antigen-presenting cell as broadly described above is also useful for producing antigen-specific regulatory T lymphocytes for suppression of an immune response to that antigen.
- the invention provides a method for producing T lymphocytes that exhibit anergy for a target antigen, comprising contacting a population of T lymphocytes, or their precursors, with an antigen-specific antigen-presenting cell as broadly described above for a time and under conditions sufficient to produce said anergic T lymphocytes.
- the antigen-specific antigen-presenting cell and T lymphocytes as broadly defined above are especially useful for inducing a tolerogenic response including the induction of an anergic response, or the suppression of a future or existing immune response, to a specified antigen or group of antigens.
- the antigen-specific immune response includes, but is not limited to, a response mediated by T lymphocytes such as cytotoxic T lymphocytes (CTLs) and T helper lymphocytes.
- CTLs cytotoxic T lymphocytes
- the antigen-specificity may be to an antigen selected from a protein antigen, a particulate antigen, an alloantigen, an autoantigen, an allergen, a bacterial antigen, a viral antigen or a parasitic antigen or immune complex.
- the invention embraces a method for modulating the immune response to an antigen, comprising administering to a patient in need of such treatment one or both of an antigen-specific antigen-presenting cell as broadly described above and an anergic T lymphocyte as broadly described above for a time and under conditions sufficient to modulate said immune response.
- the invention extends to the use of the aforesaid antigen-specific antigen-presenting cell and/or said anergic T lymphocyte as broadly described above in methods for inducing an anergic response, or for treating or preventing an allergy or an autoimmune disease, or for treating or preventing transplant rejection in a patient, by administering to a patient in need thereof an effective amount of one or both of an antigen-specific antigen-presenting cell as broadly described above and an anergic T lymphocyte as broadly described.
- the invention encompasses a method for treatment and/or prophylaxis of a disease or condition whose symptoms or aetiology are associated with the presence of an immune response, comprising administering to a patient in need of such treatment or prophylaxis an effective amount of one or both of an antigen-specific antigen-presenting cell as broadly described above and an anergic T lymphocyte 'as broadly described.
- the invention contemplates the use of an antigen-presenting cell or an antigen-specific antigen-presenting cell as broadly described above or an anergic T lymphocyte as broadly described above in the preparation of a medicament for the modulation of an immune.
- the invention also encompasses the use of an antigen-presenting cell or an antigen-specific antigen-presenting cell as broadly described above or an anergic T lymphocyte as broadly described in the study and modulation of immune responses.
- FIG 1 is a graphical representation showing suppression of primed immune responses by RelB deficient bone marrow-derived dendritic cells (BMDCs).
- BMDCs were generated from wild-type or RelB " " mice and cell surface markers were analysed by flow cytometry (a). Wild type mice were injected with BMDCs or methylated bovine serum albumin (mBSA) in complete Freund's adjuvant (CFA) as shown, and draining lymph node (DLN) mBSA-specific T cell proliferation was examined 7 days later (b). Wild type mice were injected with BMDCs or saline as shown, 7 days before or after immunization with keyhole limpet haemocyanin (KLH) in CFA (c).
- KLH keyhole limpet haemocyanin
- FIG. 2 is a diagrammatic and graphical representation showing that dendritic (DC) differentiation in the presence of an inhibitor of NF- ⁇ B translocation inhibits CD40 expression and APC function.
- BMDCs were generated in the presence or absence of BAY 11-7082.
- Nuclear and cytoplasmic extracts were immunoblotted for NF- ⁇ B subunits as shown (a).
- Cell surface marker expression was analysed by flow cytometry (b). Na ⁇ ve C57BL/6 mice were injected subcutaneously (s.c.) as shown.
- DLN T cell proliferation in vitro in response to exogenous mBSA is displayed as mean ⁇ SEM ⁇ CPM of triplicates of 5 mice assayed individually (c). Results are representative of three separate experiments.
- Figure 3 is a graphical representation showing suppression of primed immune responses by inhibition of RelB function of DCs.
- Mice were injected s.c. with BMDCs as shown, 7 d before (a) or after (b) priming with mBSA in CFA. 5 days later, mice were individually tested for DLN antigen-specific T cell proliferative, serum antibody and ear DTH responses. 7 days after s.c. or intravenous (i.v.) immunisation with varying doses of mBSA-pulsed BAY-treated BMDCs or no DCs, mice were injected with mBSA in CFA (c) or KLH in CFA (d).
- Figure 4 is a graphical representation showing suppression of primed responses by DCs correlates with RelB nuclear binding activity.
- BMDCs were generated in either GM-CSF and IL-4
- mice were primed with KLH in CFA and 7 days later injected with DCs or saline as shown. KLH-specific T cell proliferative responses were measured after 5 days (b). Nuclear extracts from DCs were bound to ELISA plates coated with NF- ⁇ B consensus oligonucleotides, and detected with either anti-RelB or anti-p50 (c).
- FIG. 5 is a graphical representation showing CD40 deficiency is sufficient to confer suppression of immunity by DCs.
- BMDCs were generated from CD40 "y" H-2 d mice in the presence or absence or BAY and cell surface markers were analysed by flow cytometry (a). Mice were injected with BMDCs or saline as shown, 7 days after immunisation with KLH in CFA. DLN KLH-specific T cell proliferation (b), and ear KLH-specific DTH responses (c) are shown. Mean ⁇ SEM cpm from groups of 5 mice tested individually are shown.
- Figure 6 is a graphical representation showing antigen-specific tolerance is "infectious”. Mice were injected with 5xl0 5 BMDCs as shown. Spleens were collected after 7 days and 5xl0 5 CD4 + CD3 + or CD4 " CD3 + cells sorted from nylon wool purified preparations were injected i.v. into non-irradiated KLH or ovalbumin (OVA)-primed syngeneic mice.
- OVA ovalbumin
- FIG. 7 is a graphical representation showing that nuclear extracts from BAY-treated monocyte-derived DC (MDDC) lack RelB-DNA binding and are unresponsive to LPS.
- MDDC were generated for 48 h from PB monocytes in the presence of GM-CSF and IL-4 in the presence (BAY DC) or absence (control DC) of 8 ⁇ M BAY, then stimulated with 1 /xg/mL LPS for 24 hr.
- Nuclear extracts were prepared and analysed for NFkB DNA binding by ELISA.
- Figure 8 is a graphical representation showing that modified human DC lack CD40 compared to immature DC.
- MDDC were generated for 48 h from PB monocytes in the presence of GM-CSF and IL-4 in the presence (modified DC) or absence (immature DC) of BAY, then stained for various cell surface markers and analysed by flow cytometry.
- Figure 9 is a graphical representation showing that modified human DC are unresponsive to CD40L.
- MDDC were generated for 48 h from PB monocytes in the presence of GM-CSF and
- IL-4 in the presence (modified DC) or absence (immature DC) of BAY, washed then incubated with or without 50 ng/ml soluble CD40L. Cells were then stained for CD86 and HLA-DR and analysed by flow cytometry.
- Figure 10 is a graphical representation showing that T cells fail to proliferate when stimulated by modified DC.
- MDDC were generated for 72 h from PB monocytes in the presence of GM-CSF and IL-4 in the presence (modified DC) or absence (immature DC) of BAY, or 48 h old immature DC were incubated for the final 24 h with 500 ng/ml LPS (mature DC), washed, then incubated with purified resting allogeneic T cells (Figure 10A), or with purified resting autologous T cells in the presence or absence of tetanus toxoid or hepatitis B surface antigen particles (Figure 10B). T cell proliferation was assessed in each case by [ 3 H] thymidine incorporation after a total of 5 days.
- Figure 11 is a graphical representation showing that T cells remain viable when stimulated by modified DC.
- MDDC were generated for 72 h from PB monocytes in the presence of GM-CSF and IL-4 in the presence (modified DC) or absence (immature DC) of BAY, or 48 h old immature DC were incubated for the final 24 h with 500ng/ml LPS (mature DC), washed, then incubated with purified resting allogeneic T cells for a total of 10 days. Control T cells were incubated in the absence of DC. Viability was measured flow cytometrically by the % cells unstained after propidium iodide addition.
- Figure 12 is a graphical representation showing that modified DC do not induce IFN ⁇ production by T cells.
- MDDC were generated for 48 h from PB monocytes in the presence of GM- CSF and IL-4 in the presence (modified DC) or absence (immature DC) of BAY, washed, then incubated with purified resting allogeneic T cells. Interferon- ⁇ was assayed 3 days later in the culture supematants by ELISA.
- Figure 13 is a graphical representation showing that the level of CD40 expression by DC correlates with the T cell proliferative response in MLR.
- MDDC were generated for 48 h from PB monocytes in the presence of GM-CSF and IL-4 in the presence of varying concentrations of BAY, and the expression of CD40 was determined by flow cytometry. DC from each culture were also washed then incubated with purified resting allogeneic T cells. T cell proliferation was assessed by [ 3 H] thymidine incorporation after a total of 5 days. % CD40 + DC and [ 3 H] thymidine uptake in response to stimulation of 1 x 10 5 allogeneic T cells by 5 x 10 3 DC is shown for each DC culture. Regression line with 95% confidence interval curves is shown. Individual data points show triplicate means + SEM.
- FIG 14 is a graphical representation showing that modified DC pulsed with arthritogenic antigen suppress antigen-induced arthritis in mice after clinical disease onset.
- Mono- articular antigen-induced arthritis (AIA) was induced in male C57/B16 mice as previously described (van den Berg W et al, 1982). Briefly, on day -8, mice were primed subcutaneously in each axilla with 100 mg mBSA, complete Freunds adjuvant and 2% Tween 80, and 400 ng pertussis toxin were injected intra-peritoneally. On day 0, 60 mg mBSA in 10ml saline were injected into one knee and 10ml saline into the contra-lateral knee, under anaesthesia.
- AIA Mono- articular antigen-induced arthritis
- Murine DC were generated for 7 days from bone marrow precursors in serum free medium, in the presence of 10 ng/ml GM-CSF, 10 ng/ml IL-4 and 2 ⁇ M BAYl 1-7082, pulsed with mBSA, then washed. Mice were injected s.c. into the tail base with 0.5 x 10 6 DC either 2, 4 or 6 days after the knee joint injections, or left untreated (AIA control). Joints were graded on alternate days for severity using a semi-quantitative scale from 0 (normal) to 5 (severe) based on the degree of swelling, measured by callipers under anaesthesia (graph shows mean ⁇ SEM). The mean swelling score of the control joints was 0 (data not shown).
- Figure 15 is a graphical representation showing that suppression of AIA by modified DC is antigen-specific.
- ALA was induced as described for Figure 14.
- Murine DC were generated for 7 days from bone marrow precursors in serum free medium, in the presence of 10 ng/ml GM-CSF, 10 ng/ml IL-4 and in the presence or absence of 2 ⁇ M BAYl 1-7082, pulsed with either mBSA or KLH, then washed. Mice were injected s.c. into the tail base with 0.5 x 10 6 DC either 2, 4 or 6 days after the knee joint injections, or left untreated (AIA control).
- FIG. 16 is a graphical representation showing treatment of full blown inflammatory arthritis with NFkB " DC is equivalent to TNF ⁇ neutralization.
- Mono-articular antigen-induced arthritis (AIA) was induced in male C57/B16 mice as described for Figure 14.
- Murine DC were generated for 7 days from bone marrow precursors in serum free medium, in the presence of 10 ng/ml GM-CSF, 10 ng/ml IL-4 and 7 ⁇ M BAYl 1-7082, pulsed with mBSA, then washed.
- mice were injected s.c. into the tail base with 0.5 x 10 6 DC 6 days after the knee joint injections, or i.p. with 100 ⁇ g/mL anti-TNF ⁇ , both DC and anti-TNF ⁇ , or left untreated (AIA control). 14 days after knee joint injection of mBSA, the same joints were challenged with lOOU IL-l ⁇ . 2 days later mice were either treated with 0.5 x 10 6 DC or irrelevant antibody. Joints were graded on alternate days for severity using a semi-quantitative scale from 0 (normal) to 5 (severe) based on the degree of swelling, measured by callipers under anesthesia (graph shows mean ⁇ SEM). The mean swelling of the control joints was 0 (data not shown).
- Figure 17 is a graphical representation showing alteration in antigen-specific antibody isotype in arthritic mice treated with NFkB " DC.
- Mono-articular antigen-induced arthritis (AIA) was induced in male C57/B16 mice as described for Figure 16.
- Murine DC were generated for 7 days from bone marrow precursors in serum free medium, in the presence of 10 ng/mL GM-CSF, 10 ng/mL IL-4 and 7 ⁇ M BAYl 1-7082, pulsed with mBSA, then washed. Mice were injected s.c. into the tail base with 0.5 x 10 6 DC 6 days after the knee joint injections or left untreated (ALA control).
- mice 14 days after knee joint injection of mBSA, the same joints were challenged with 100U IL-l ⁇ . 2 days later mice previously treated were retreated with 0.5 x 10 6 DC (clinical scores shown in Figure 17). mBSA-specific antibodies in sera were analyzed by ELISA on day 20 and typed with isotype-specific secondary antibodies.
- Figure 18 is a graphical representation illustrating long term suppression of antigen- specific responses by NFkB " DC.
- Mice were primed with antigen KLH in complete Freund's adjuvant. 7 days later, groups of 5 mice were treated with either 5 x 10 5 bone marrow derived DC generated in the presence (Bay DC) or absence of BAYl 1-7082 and exposed or not to the antigen KLH, or with saline. A final group of mice was left unprimed and untreated. KLH-specific delayed type hypersensitivity was tested at 1, 2, 3 and 8 months in each mouse.
- Figure 19 is a graphical representation showing the restoration of suppression following boosting with antigen.
- mice were boosted with KLH and complete Freund's adjuvant 1 week after the 3-month DTH analysis
- mice were boosted with KLH and complete Freund's adjuvant 1 week after the 1- and 3-month DTH analyses.
- Figure 20 is a graphical representation showing that administration of NFkB- donor or recipient DC at the time of allogeneic bone marrow transplantation reduces GVHD mortality.
- Figure 21 is a graphical representation showing the prevention of type I diabetes in NOD mice by NF- ⁇ B " dendritic cells.
- Murine DC were generated for 7 days from NOD or proinsulin- NOD bone marrow precursors in the presence of GM-CSF, IL-4 and BAYl 1-7082, then washed. 12 mice per group were injected s.c. into the tail base with 0.5 x 10 6 DC or PBS at the age of 4 weeks or left untreated. Mice were tested weekly for presence of glycosuria. Glycosuric mice were tested by glucometer for blood glucose. Diabetes was diagnosed if blood glucose was greater than 12, on 2 consecutive occasions.
- Figure 22 is a graphical representation showing the phenotype of NF- ⁇ B " dendritic cells is reproducible with different NF- ⁇ B inhibitors.
- Dendritic cells were generated from human monocytes for 48 hours in the presence of GM-CSF, IL-4 and in the presence or absence of either 10 ⁇ M BAYl 1-7082 or 2.5 or 10 ⁇ M PSI (N-benzyloxycarbonyl-rie-Glu(O-tert-butyl)-Ala- leucinal). Cells were then stained with anti-CD40, anti-CD86, anti-HLA-DR and anti-MHC class I niAb (green lines) and compared with isotype control staining (purple) by flow cytometry.
- Figure 23 is a graphical representation showing that bone marrow macrophages also induce antigen-specific tolerance.
- Dendritic cells or macrophages were generated from bone marrow precursors in the presence of growth factors and 10 ⁇ M Bay 11-7082 then washed and exposed to the antigen KLH.
- Recipient mice (5 per group) were primed with KLH in CFA, then administered 5x10 5 DC or macrophages subcutaneously, or no treatment.
- One group was not primed with KLH in CFA.
- KLH-specific delayed type hypersensitivity responses were measured on all groups 4 days after administration of antigen presenting cells.
- an element means one element or more than one element.
- lymphocytes and B lymphocytes are anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.
- antigen is meant all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, preferably a mammal. Such antigens are also reactive with antibodies from animals immunised with said protein, peptide, or other molecule or macromolecule.
- antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
- a level and/or functional activity in the context of a protein produced by a specified cell is to be taken in its broadest sense and includes a level and/or functional activity of the protein that is produced in a single cell or in a plurality or population of cells. In the latter case, therefore, it will be understood that the phrase will encompass a mean level and/or functional activity of the protein produced by a plurality or population of cells.
- autologous is meant something (e.g., cells, tissues etc) derived from the same organism.
- allogeneic refers to cells, tissues, organisms etc that are of different genetic constitution.
- culture refers to the set of procedures used in vitro where a population of cells (or a single cell) is incubated under conditions which have been shown to support the growth or maintenance of the cells in vitro.
- the art recognises a wide number of formats, media, temperature ranges, gas concentrations etc. which need to be defined in a culture system. The parameters will vary based on the format selected and the specific needs of the individual who practices the methods herein disclosed. However, it is recognised that the determination of culture parameters is routine in nature.
- an effective amount in the context of modulating an immune response or treating or preventing a disease or condition, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that modulation, treatment or prevention.
- the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
- expression vector any autonomous genetic element capable of directing the synthesis of a protein encoded by the vector. Such expression vectors are known by practitioners in the art.
- the term “gene” is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.
- immuno-interactive includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
- isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
- modulating is meant increasing or decreasing, either directly or indirectly, the immune response of an individual.
- operably connected or “operably linked” as used herein means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene.
- the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.
- patient refers to patients of mammalian, especially human, or other animal origin and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that “patient” does not imply that symptoms are present.
- Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, reptiles, avians, fish).
- pharmaceutically-acceptable carrier is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.
- polynucleotide or “nucleic acid' as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
- the term typically refers to oligonucleotides greater than 30 nucleotides in length.
- Polypeptide , “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non- naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
- promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue- specific or cell-type-specific manner.
- a promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates.
- the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
- Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell, and/or to alter the timing of expression of a structural gene to which it is operably connected.
- the term "recombinant polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature.
- the recombinant polynucleotide may be in the form of an expression vector.
- expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.
- recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.
- reporter lymphocyte is meant a lymphocyte which is involved in controlling responses and actions of other cells, especially of other immune cells such as B lymphocytes and T helper lymphocytes.
- reporter molecule as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a complex comprising an antigen-binding molecule and its target antigen.
- reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
- vector is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned.
- a vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
- the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
- the vector may contain any means for assuring self-replication.
- the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
- a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
- the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
- the vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
- the present invention is predicated in part on the determination that an antigen-presenting cell that produces CD40, or its equivalent, at a level and/or functional activity that is lower than that produced by an activated dendritic cell, is a potent modulator of immune responses and is especially useful not only for preventing priming of immunity but also for suppressing a previously primed immune response to a specified antigen or group of antigens.
- the antigen-presenting cell cannot be induced to express CD40, or its equivalent, at an equivalent level and/or functional activity as that produced by an activated antigen presenting cell.
- the antigen-presenting cell cannot be induced to express CD40, or its equivalent, at a higher level and/or functional activity than that produced by an immature antigen-presenting cell.
- the antigen-presenting cell is other than a B lymphocyte.
- the antigen-presenting cell produces CD40, or its equivalent, at a level and/or functional activity that is less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, of that produced by a mature or activated dendritic cell.
- the antigen-presenting cell produces CD40, or its equivalent, at a level and/or functional activity that is less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, of that produced by a mature or activated dendritic cell.
- the antigen-presenting cell produces CD40, or its equivalent, at a level and/or functional activity that is less than that produced by an immature dendritic cell.
- the antigen-presenting cell is further characterised by being non proliferating; and expressing one or both of a class I and a class II MHC determinant.
- the antigen-presenting cell preferably encompasses both professional and facultative types of antigen-presenting cells.
- professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells.
- facultative antigen-presenting cells include but are not limited to activated T cells, astrocytes, follicular cells, endothelium and fibroblasts.
- the antigen-presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells.
- the antigen-presenting cell expresses CDllc and includes a dendritic cell.
- the antigen-presenting cell is preferably further characterised by producing NF-/cB or component thereof, especially RelB, at a level and/or functional activity that is less than about
- the antigen-presenting cell produces NF- ⁇ B or component thereof, especially RelB, at a level and/or functional activity that is lower than that produced by an immature dendritic cell (Thompson et al, 2002).
- the antigen-presenting cell e.g., dendritic cell
- the antigen-presenting cell cannot be induced to express NF- ⁇ B or component thereof, at a higher level and/or functional activity than an immature antigen presenting cell (e.g., an immature dendritic cell).
- the antigen-presenting cell is further characterised by expressing an immunostimulatory molecule, such as CD86 or CD80, or their equivalents.
- an immunostimulatory molecule such as CD86 or CD80, or their equivalents.
- the immunostimulatory molecule is CD86 or its equivalent, and is suitably produced at a level and/or functional activity which is suitably at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, of that produced by an activated dendritic cell.
- the antigen-presenting cell is further characterised by presenting an antigen, which is preferably in the context of MHC molecules expressed by the cell, and causing T lymphocytes to exhibit anergy to that antigen by contact of the cell with the lymphocytes.
- the present invention also contemplates an isolated and preferably purified population of antigen-presenting cells as broadly described above.
- Numerous techniques are known to practitioners in the art for isolating and/or purifying cellular populations, including the use of surgical removal of tissue, flow cytometry techniques such as fluorescence-activated cell sorting (FACS), immunoaffinity separation (e.g., magnetic bead separation such as DynabeadTM separation), density separation (e.g., metrizamide, PercollTM, or FicollTM gradient centrifugation), and cell-type specific density separation.
- FACS fluorescence-activated cell sorting
- immunoaffinity separation e.g., magnetic bead separation such as DynabeadTM separation
- density separation e.g., metrizamide, PercollTM, or FicollTM gradient centrifugation
- cell-type specific density separation e.g., cell-type specific density separation.
- an antigen-presenting cell according to the present invention is obtained by contacting a precursor of the antigen-presenting cell with an NF- ⁇ B inhibitor for a time and under conditions sufficient to differentiate an antigen-presenting cell from the precursor and to inhibit or otherwise reduce the level and/or functional activity of NF- ⁇ B in said cell.
- the antigen-presenting cell of the invention can be produced by contacting an antigen-presenting cell, or its precursor, with an inhibitor of CD40, or its equivalent, for a time and under conditions sufficient to produce a modified antigen-presenting cell that produces CD40, or its equivalent, at a level and/or functional activity that is reduced or abrogated relative to that of said antigen- presenting cell or its precursor.
- the antigen-presenting cell, or its precursor, or the modified antigen-presenting cell is contacted with an agent that increases the level and/or functional activity of an immunostimulatory molecule, such as CD86 or CD80, or their equivalents (e.g., a polynucleotide from which the immunostimulatory molecule can be expressed), for a time and under conditions sufficient to enhance or otherwise elevate the level and/or functional activity of the immunostimulatory molecule.
- an immunostimulatory molecule such as CD86 or CD80, or their equivalents (e.g., a polynucleotide from which the immunostimulatory molecule can be expressed)
- Antigen-presenting cell precursors can be isolated by methods known to those of skill in the art.
- the source of precursor will differ depending upon the antigen-presenting cell required for modulating a specified immune response.
- the antigen-presentmg cell can be selected from include dendritic cells, macrophages, monocytes and other cells of myeloid lineage.
- precursors of antigen-presenting cells can be isolated from any tissue, but are most easily isolated from blood, cord blood or bone marrow (Sorg et al, 2001; Zheng et al, 2000).
- rheumatoid synovial tissue or fluid following biopsy or joint tap rheumatoid synovial tissue or fluid following biopsy or joint tap
- Other examples include, but are not limited to liver, spleen, heart, kidney, gut and tonsil (Lu et al, 1994; Mcllroy et al, 2001; Vremec et al, 2000) (Hart and Fabre, 1981; Hart and McKenzie, 1988; Pavli et al, 1990).
- Leukocytes isolated directly from tissue provide a major source of antigen-presenting cell precursors. Typically, these precursors can only differentiate into antigen-presenting cells by culturing in the presence or absence of various growth factors. According to the practice of the present invention, the antigen-presenting cells may be so differentiated from crude mixtures or from partially or substantially purified preparations of precursors. Leukocytes can be conveniently purified from blood or bone marrow by density gradient centrifugation using, for example, Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs), or by ammonium chloride lysis of red cells (leukocytes or white blood cells).
- Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs)
- ammonium chloride lysis of red cells leukocytes or white blood cells.
- Tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g., basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers.
- tissue dendritic cells e.g., basal layer of epidermis
- Langerhans cell precursors express CD1 molecules as well as HLA-DR and can be purified on this basis.
- the antigen-presenting cell precursor is a precursor of macrophages.
- these precursors can be obtained from monocytes of any source and can be differentiated into macrophages by prolonged incubation in the presence of medium and macrophage colony stimulating factor (M-CSF) (Erickson-Miller et al, 1990; Metcalf and Burgess, 1982).
- M-CSF medium and macrophage colony stimulating factor
- the antigen presenting cell precursor is a precursor of Langerhans cells.
- Langerhans cells can be generated from human monocytes or CD34 + bone marrow precursors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), JL- 4/TNF ⁇ and TGF ⁇ (Geissmann et al, 1998; Strobl et al, 1997a; Strobl et al, 1997b; Strobl et al, 1996)
- GM-CSF granulocyte/macrophage colony-stimulating factor
- JL- 4/TNF ⁇ JL- 4/TNF ⁇
- TGF ⁇ granulocyte/macrophage colony-stimulating factor
- the antigen-presenting cell precursor is a precursor of dendritic cells.
- dendritic cell precursors can be obtained from peripheral blood, cord blood or bone marrow. These include monocytes, CD34 + stem cells, granulocytes, CD33 + CD1 lc + DC precursors, and committed myeloid progenitors - described below.
- Monocytes can be purified by adherence to plastic for 1-2 h in the presence of tissue culture medium (e.g., RPMI) and serum (e.g., human or foetal calf serum), or in serum- free medium (Anton et al, 1998; Araki et al, 2001; Mackensen et al, 2000; Nestle et al, 1998; Romani et al, 1996; Thurner et al, 1999). Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001).
- tissue culture medium e.g., RPMI
- serum e.g., human or foetal calf serum
- Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001).
- Monocytes can also be purified by immunoaffinity techniques, including immunomagnetic selection, flow cytometric sorting or panning (Araki et al, 2001; Battye and Shortman, 1991), with anti-CD14 antibodies to obtain CDM 1 " cells.
- the numbers (and therefore yield) of circulating monocytes can be enhanced by the in vivo use of various cytokines including GM-CSF (Groopman et al, 1987; Hill et al, 1995).
- Monocytes can be differentiated into dendritic cells by prolonged incubation in the presence of GM-CSF and IL-4 (Romani et al, 1994; Romani et al, 1996).
- a combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/mL, more preferably between about 500 to about 1000 U/mL and even more preferably between about 800 U/mL (GM-CSF) and 1000 U/mL (IL-4) produces significant quantities of immature dendritic cells, i.e., antigen-capturing phagocytic dendritic cells.
- Other cytokines which promote differentiation of monocytes into antigen-capturing phagocytic dendritic cells include, for example, IL-13.
- CD34 + stem cells can also be generated from CD34 + bone marrow derived precursors in the presence of GM-CSF, TNF ⁇ ⁇ stem cell factor (SCF, c-kitL), or GM- CSF, IL-4 ⁇ flt3L (Bai et al, 2002; Chen et al, 2001; Loudovaris et al, 2001).
- CD34 + cells can be derived from a bone marrow aspirate or from blood and can be enriched as for monocytes using, for example, immunomagnetic selection or immunocolumns (Davis et al, 1994).
- CD34 + cells in blood can be enhanced by the in vivo use of various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001; Pulendran et al, 2000; Robinson et al, 2000).
- various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001; Pulendran et al, 2000; Robinson et al, 2000).
- DC can be generated from committed early myeloid progenitors in a similar fashion to CD34 + stem cells, in the presence of GM-CSF and IL-4/TNF.
- myeloid precursors infiltrate many tissues in inflammation, including rheumatoid arthritis synovial fluid (Santiago-Schwarz et al, 2001).
- Expansion of total body myeloid cells including circulating dendritic cell precursors and monocytes can be achieved with certain cytokines, including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro-GP) (Fleming et al, 2001; Pulendran et al, 2000; Robinson et al, 2000).
- Dendritic cells can also be generated from peripheral blood neutrophil precursors in the presence of GM- CSF, IL-4 and TNF ⁇ (Kelly et al, 2001; Oehler et al, 1998). It should be noted that dendritic cells can also be generated, using similar methods, from acute myeloid leukemia cells (Oehler et al, 2000). Tissue DC precursors and other sources of APC precursors.
- Transformed or immortalised dendritic cell lines may be produced using oncogenes such as v-myc as for example described by (Paglia et al, 1993) or by myb (Banyer and Hapel, 1999; Gonda et al, 1993). Circulating DC precursors. These have been described in human and mouse peripheral blood. One can also take advantage of particular cell surface markers for identifying suitable dendritic cell precursors.
- various populations of dendritic cell precursors can be identified in blood by the expression of CD1 lc and the absence or low expression of CD14, CD19, CD56 and CD3 (ODoherty et al, 1994; O'Doherty et al, 1993). These cells can also be identified by the cell surface markers CD13 and CD33 (Thomas et al, 1993b).
- a second subset which lacks CD 14, CD 19, CD56 and CD3, known as plasmacytoid dendritic cell precursors, does not express CD lie, but does express CD 123 (IL-3R chain) and HLA-DR (Farkas et al, 2001; Grouard et al, 1997; Rissoan et al, 1999).
- CDllc + dendritic cell precursors are HLA-DR 1" , however some precursors may be HLA-DR-.
- MHC class II expression has been clearly demonstrated for peripheral blood dendritic cell precursors (del Hoyo et al, 2002).
- CD33 + CD14 " ⁇ o or CDllc + HLA-DR + , lineage marker-negative dendritic cell precursors described above can be differentiated into more mature antigen-presenting cells by incubation for 18-36 h in culture medium or in monocyte conditioned medium (Thomas et al, 1993b; Thomas and Lipsky, 1994) (O'Doherty et al, 1993).
- peripheral blood dendritic cells are characterised by low density and so can be purified on density gradients, including metrizamide and Nycodenz (Freudenthal and Steinman, 1990; Vremec and Shortman, 1997), or by specific monoclonal antibodies, such as but not limited to the CMRF-44 mAb (Fearnley et al, 1999; Vuckovic et al, 1998).
- Plasmacytoid dendritic cells can be purified directly from peripheral blood on the basis of cell surface markers, and then incubated in the presence of IL-3 (Grouard et al, 1997; Rissoan et al, 1999).
- plasmacytoid DC can be derived from density gradients or CMRF-44 selection of incubated peripheral blood cells as above.
- dendritic cells generated from any precursor when incubated in the presence of activation factors such as monocyte-derived cytokines, lipopolysaccharide and DNA containing CpG repeats, cytokines such as TNF-o; IL-6, IFN-c ⁇ IL-l ⁇ necrotic cells, readherence, whole bacteria, membrane components, RNA or polylC, immature dendritic cells will become activated (Clark, 2002; Ralpher et al, 2002; Kaisho and Akira, 2002; Koski et al, 2001). This process of dendritic cell activation is inhibited in the presence of NF-/ B inhibitors (O'Sullivan and Thomas, 2002).
- activation factors such as monocyte-derived cytokines, lipopolysaccharide and DNA containing CpG repeats
- cytokines such as TNF-o; IL-6, IFN-c ⁇ IL-l ⁇ necrotic cells, readherence, whole bacteria, membrane components, RNA or
- an antigen-presenting cell of the present invention is produced by contacting a precursor of an antigen-presenting cell, such as but not limited to the precursors mentioned above, with an NF- ⁇ B inhibitor.
- an NF- ⁇ B inhibitor Numerous inhibitors of NF- ⁇ B are known to those of skill in the art. The inhibitors may act directly on NF-KB, or indirectly via another entity that regulates the level and/or functional activity of NF- ⁇ B. Indirect inhibitors include, but are not limited to, inhibitors of proteolysis and inhibitors of nuclear translocation of NF- ⁇ B.
- the inhibitor of nuclear translocation of NF- ⁇ B includes, but is not limited to, deoxyspergualin (Tapper, et al, 1995, J Immunol. 155:2427-2436) or derivatives or analogues of deoxyspergualin including, for example, mefhyl-deoxyspergualin, a deoxyspergualin analog lacking a chiral centre (e.g., LF 08- 0299) (Andoins et al, 1996, Transplantation 62:1543-1549) and the derivatives or analogues identified in US Pat. No. 4,518,532, US Pat. No. 4,518,532, US Pat. No. 4,252,299, US Pat. No.
- the inhibitor of proteolysis is preferably but not exclusively a proteosome inhibitor such as PSI (Traechner, et al, 1994, EMBO J. 13:5433-5441; Griscavage, et al, 1996, Proc. Natl. Acad. Sci. USA 93:3308-3312; Bondeson, et al, 1999, J.
- PSI Proteinosome inhibitor
- Immunol 162:2939-2945 ALLN (Jobin, et al, 1998, Hepatology 27:1285-1295), lactacystin (Delic, et al, 1998, Br. J. Cancer 77:1103-1107), MG-132 (Jobin, et al. supra), C-LFF and calpain inhibitors (Neauparfant and Hiscott, 1996, Cytokine & Growth Factor Reviews 7:175- 190) and CVT-134 (Lum, et al, 1998, Biochem. Pharmacol 55:1391-1397).
- Other indirect NF- ⁇ B inhibitors include: caffeic acid phenethyl ester (Natarajan, et al, 1992, Proc.
- the indirect inhibitor of NF- ⁇ B is an inhibitor of I ⁇ B degradation including, but not limited to, inhibitors of I ⁇ B phosphorylation, I ⁇ B ubiquitination and proteolytic degradation of I ⁇ B, for example, by the proteosome.
- the NF- ⁇ B inhibitor is a direct inhibitor of NF- ⁇ B, i.e., acts directly on the level (quantity), cellular location or activity of NF- ⁇ B.
- the inhibitor may be a naturally occurring regulator of NF- ⁇ B that interacts directly with NF- ⁇ B, such as an I B, especially I ⁇ B ⁇ , as for example described by Makarov (1997, Gene Therapy 4:846-852) and in PCT/GB98/02753.
- I B especially I ⁇ B ⁇
- Bondeson et al. (1999, Proc. Natl. Acad. Sci. USA 96:5668-5673 describe an I ⁇ B-encoding adenovirus.
- inhibitors of NF- ⁇ B include inhibitors of nuclear localisation of NF- ⁇ B, inhibitors of DNA binding of NF- ⁇ B as well as antisense nucleic acid molecules or oligonucleotides, which are complementary or encode at least a portion of any of the NF- ⁇ B subunits, e.g., p50, p65, RelB.
- the inhibitor of NF- ⁇ B is an inhibitor of RelB or p50.
- Such an inhibitor may be a ribozyme which selectively destroys RNA encoding NF-/ B, or an antisense molecule which prevents transcription of NF- ⁇ B or an antigen-binding molecule (e.g., antibody, Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments or other synthetic antigen-binding molecules such as synthetic stabilised Fv fragments, dAbs, minibodies and the like) which blocks NF-KB action (O'Sullivan et al, 2000).
- an antigen-binding molecule e.g., antibody, Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments or other synthetic antigen-binding molecules such as synthetic stabilised Fv fragments, dAbs, minibodies and the like
- Especially preferred inhibitors of NF- ⁇ B are inhibitors of I ⁇ B phosphorylation, such as BAY 11-7082 (BioMol, Plymouth Meeting, PA). BAY 11-7082 has been shown to block TNF-cc- stimulated NF- ⁇ B translocation through inhibition of I B phosphorylation (Pierce et al, 1997).
- an antigen-presenting cell of the present invention is produced by contacting a differentiated antigen-presenting cell, or a precursor thereof, as for example described in Section 2.1, with a CD40 inhibitor for a time and under conditions sufficient to produce a modified antigen-presenting cell that produces CD40, or its equivalent, at a reduced or abrogated level and/or functional activity relative to that of said antigen-presenting cell or its precursor.
- the CD40 inhibitor can be an indirect inhibitor but is preferably a direct inhibitor of CD40, i.e., acts directly on the level (quantity), cellular location or activity of CD40.
- the differentiated antigen-presenting cell, or a precursor thereof, as for example described in Section 2.1, or the modified antigen-presenting cell as described above is further contacted with an activator or inducer of an immunostimulatory molecule, especially of CD86 or CD80, or their equivalents, for a time and under conditions sufficient to enhance or otherwise elevate the level and/or functional activity of the immunostimulatory molecule.
- the activator or inducer can be a transcriptional activator, which enhances the expression of the immunostimulatory molecule, or an expression vector from which the immunostimulatory molecule is expressible.
- the amount of modulator (e.g., NF-/cB inhibitor) to be placed in contact with the antigen- presenting cell precursors can be determined empirically by persons of skill in the art.
- the precursor is cultured with an NF- ⁇ B inhibitor for the duration of the process of dendritic cell differentiation from its precursors, typically for about 1 to 120 hours, preferably for about 4 to 36 hours for peripheral blood dendritic cell precursors other than monocytes and tissue precursors, and preferably between about 48 to 120 hours and up to 168 hours for monocyte precursors, and greater than 108 hours (7-10 days) for CD34 + and other committed myeloid precursors.
- cells and inhibitors are incubated in the presence of one or more factors required for the differentiation of the precursor to the antigen-presenting cell of interest.
- dendritic cell precursors which are preferably derived from bone marrow, are cultured in the presence of dendritic cell growth factors and an NFKB inhibitor, especially BAY 11-7082, for about 6-10 days, or monocyte precursors which are preferably derived from peripheral blood are incubated in the presence of dendritic cell growth factors and an NFKB inhibitor, especially BAY 11-7082, for about 2-5 days.
- an NFKB inhibitor especially BAY 11-7082
- the antigen-presenting cells of the present invention are useful for modulating an immune response, and are especially useful for inducing a tolerogenic response including the induction of an anergic response, and the suppression of a future or existing immune response, to one or more target antigens.
- Antigen-specific antigen-presenting cells can be produced by contacting an antigen-presenting cell of the invention with at least one antigen that corresponds to a specified target antigen, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen-presenting cell.
- a precursor of the antigen-presenting cell can be co-cultured with an NF- ⁇ B inhibitor, together with the antigen, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for an antigen-presenting cell to differentiate from the precursor and for the level and/or functional activity of NF- ⁇ B in the antigen- presenting cell to be abrogated or otherwise reduced and for the antigen, or processed form thereof, to be presented by the antigen-presenting cell.
- a variety of possible antigens exist for which modulation of an immune response, especially the induction of a tolerogenic immune response, and more especially the induction of antigen-specific lymphocyte anergy, may be important.
- antigens for which such modulation of an immune response may be important include soluble antigens, e.g., soluble proteins or fragments of insoluble complexes, particulate antigens, e.g., bacteria or parasites, and allergens.
- exemplary antigen which may be used in the practice of the present invention include, but are not limited to, self antigens that are targets of autoimmune responses, allergens and transplantation antigens.
- self antigens include, but are not restricted to, lupus autoantigen, Smith, Ro, La, Ul- RNP, fibrillin (scleroderma), GAD65 (diabetes related), insulin, myelin basic protein, histones, PLP, collagen, glucose-6-phosphate isomerase, citrullinated proteins and peptides, thyroglobulin, various fRNA synthetases, acetyl choline receptor (AchR), MOG, proteinase-3, myeloperoxidase etc.
- allergens include, but are not limited to, Fel d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571), Der p I, Der p II, Der fl or Der fll (i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281).
- Fel d 1 i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571
- Der p I, Der p II, Der fl or Der fll i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281).
- allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens; fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chimomidae (non-biting midges); other insects such as the housefly, fruitfly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein detergent additives.
- the antigen(s) may be isolated from a natural source or may be prepared by recombinant techniques as is known in the art.
- peptide antigens can be eluted from the MHC and other presenting molecules of antigen-presenting cells obtained from a cell population or tissue for which a modified immune response is desired.
- the eluted peptides can be purified using standard protein purification techniques known in the art (Rawson et al, 2000; Smithers et al, 2002). If desired, the purified peptides can be sequenced and synthetic versions of the peptides produced using standard protein synthesis techniques as for example described below.
- crude antigen preparations can be produced by isolating a sample of a cell population or tissue for which a modified immune response is desired, and either lysing the sample or subjecting the sample to conditions that will lead to the formation of apoptotic cells (e.g., irradiation with ultra violet or with gamma rays, viral infection, cytokines or by depriving cells of nutrients in the cell culture medium, incubation with hydrogen peroxide, or with drugs such as dexamethasone, ceramide chemotherapeutics and anti-hormonal agents such as Lupron or Tamoxifen).
- the lysate or the apoptotic cells can then be used as a source of crude antigen for contact with the antigen-presenting cells.
- the antigen When the antigen is known, it may be conveniently prepared in recombinant form using standard protocols as for example described in: Sambrook, et al, MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
- an antigen may be prepared by a procedure including the steps of (a) providing an expression vector from which the target antigen or analogue or mimetic thereof is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant polypeptide from the vector; and (d) isolating the recombinant polypeptide.
- the expression vector will comprise an antigen-encoding polynucleotide which is operably connected to a regulatory polynucleotide.
- the antigen-encoding polynucleotide can be constructed from any suitable parent polynucleotide that codes for an antigen that corresponds to the target antigen of interest.
- the parent polynucleotide is suitably a natural gene or portion thereof. However, it is possible that the parent polynucleotide is not naturally-occurring but has been engineered using recombinant techniques.
- the regulatory polynucleotide suitably comprises transcriptional and/or translational control sequences, which will generally be appropriate for the host cell used for expression of the antigen-encoding polynucleotide.
- the transcriptional and translational regulatory control sequences include, but are not limited to, a promoter sequence, a 5' non-coding region, a czs-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream open reading frame, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence, termination codon, translational stop site and a 3' non-translated region.
- Constitutive or inducible promoters as known in the art are contemplated by the invention.
- the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
- Promoter sequences contemplated by the present invention may be native to the host cell to be introduced or may be derived from an alternative source, where the region is functional in the host cell.
- the expression vector may also comprise a 3' non-translated sequence, which usually refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
- the polyadenylation signal is characterised by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
- the 3' non-translated regulatory DNA sequence preferably includes from about 50 to 1,000 nucleotide base pairs and may contain transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
- the expression vector further contains a selectable marker gene to allow the selection of transformed host cells.
- Selection genes are well known in the art and will vary with the host cell used.
- the expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner.
- fusion partners typically provided by the expression vector
- GST glutathione-S-transferase
- Fc portion of human IgG Fc portion of human IgG
- MBP maltose binding protein
- HIS 6 hexahistidine
- relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively.
- Many such matrices are available in "kit” form, such as the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners and the Pharmacia GST purification system.
- the fusion partners also have protease cleavage sites, such as for Factor X a or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom.
- Fusion partners also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available.
- epitope tags for which specific monoclonal antibodies are readily available include c-Myc, influenza virus haemagglutinin and FLAG tags.
- the step of introducing the expression vector into the host cell may be effected by any suitable method including transfection, transduction of viral vectors, including adenoviral, modified lentiviral and other retroviral vectors, and transformation, the choice of which will be dependent on the host cell employed. Such methods are well known to those of skill in the art.
- Recombinant polypeptides of the invention may be produced by culturing a host cell transformed with the expression vector under conditions appropriate for protein expression, which will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.
- Suitable host cells for expression may be prokaryotic or eukaryotic.
- One preferred host cell for expression of a polypeptide according to the invention is a bacterium.
- the bacterium used may be Escherichia coli.
- the host cell may be an insect cell such as, for example, SF9 cells that may be utilised with a baculovirus expression system.
- the antigen can be synthesised using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) or by Roberge et al. (1995, Science 269: 202).
- exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen- presenting cells, especially dendritic cells. These methods include insertion of antigen into pH- sensitive liposomes (Zhou and Huang, 1994, Immunomethods, 4:229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al, 1988, Cell, 54:777-785), coupling of antigens to potent adjuvants (Aichele et al, 1990, J. Exp.
- E. coli or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery.
- dendritic cells as particulate antigen, or apoptotic bodies respectively
- a delivery system might be logically combined with a substance for inhibiting ⁇ F- ⁇ B, such as a plasmid encoding dominant negative I ⁇ B ⁇ (Pai et al, 2002).
- Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et al, 1996, Eur. J. Immunol, 26(11): 2595-2600).
- an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway.
- cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 and U.S. Patent No. 6,352,697), phospholipases (see, e.g., Camilli et al, 1991, J. Exp. Med.
- pore-forming toxins e.g., an alpha-toxin
- natural cytolysins of gram-positive bacteria such as listeriolysin O (LLO, e.g., Mengaud et al, 1988, Infect. Immun. 56: 766-772 and Portnoy et al, 1992, Infect. Immun. 60: 2710-2717
- LLO listeriolysin O
- SLO srreptolysin O
- PFO perfringolysin O
- cytolysins may be advantageously used.
- listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al, Infect. Immun. 1992, 60: 2710-2717).
- the cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen-presenting cells.
- the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell.
- the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
- a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
- the delivery vehicle is non- virulent.
- the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No.
- Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc.
- nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5.alpha., etc.
- Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc.
- the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.
- the delivery vehicles described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells.
- the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and endosomes).
- antigen-presenting cells The amount of antigen to be placed in contact with antigen-presenting cells can be determined empirically by persons of skill in the art. Typically antigen-presenting cells are incubated with antigen for about 1 to 6 hr at 37° C, although it is also possible to expose antigen- presenting cells to antigen for the duration of incubation with growth factors and inhibitor. Usually, for purified antigens and peptides, 0.1-10 ⁇ g/mL is suitable for producing antigen-specific antigen- presenting cells, dendritic cells are exposed to apoptotic bodies in approximately 1:1 ratio, and bacteria (Albert et al, 1998; Corinti et al, 1999).
- the antigen should be exposed to the antigen- presenting cells for a period of time sufficient for those cells to internalise the antigen.
- the time and dose of antigen necessary for the cells to internalise and present the processed antigen may be determined using pulse-chase protocols in which exposure to antigen is followed by a washout period and exposure to a read-out system e.g., antigen reactive T cells. Once the optimal time and dose necessary for cells to express processed antigen on their surface is determined, a protocol may be used to prepare cells and antigen for inducing tolerogenic responses.
- an antigen-presenting cell may vary depending on the antigen or form of antigen employed, its dose, and the antigen- presenting cell employed, as well as the conditions under which antigen loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
- an antigen of interest can be produced inside the antigen- presenting cell by introduction of a suitable expression vector as for example described above.
- the antigen-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques.
- the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215.
- the replacement step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of a parent polynucleotide.
- the expression vector for introduction into the antigen-presenting cell will be compatible therewith such that the antigen-encoding polynucleotide is expressible by the cell.
- expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adeno virus, adeno-associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses.
- Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin. Biotecltnol 11(2):205-208), Vigna and Naldini (2000, J. Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(l):33-40), Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) and Walther and Stein (2000, Drugs 60(2):249-271).
- the expression vector is introduced into the antigen-presenting cell by any suitable means which will be dependent on the particular choice of expression vector and antigen-presenting cell employed.
- introduction can be effected by use of contacting (e.g., in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like.
- contacting e.g., in the case of viral vectors
- electroporation transformation, transduction, conjugation or triparental mating
- transfection infection membrane fusion with cationic lipids
- high-velocity bombardment with DNA-coated microprojectiles high-velocity bombardment with DNA-coated microprojectiles
- incubation with calcium phosphate-DNA precipitate direct microinjection into single cells, and the like.
- the vectors are introduced by means of cationic lipids, e.g., liposomes.
- the antigen-specific antigen-presenting cells can be obtained by isolating antigen-presenting cell precursors from a cell population or tissue to which modification of an immune response is desired and causing these precursors to differentiate into antigen- presenting cells of the invention using the methods described herein.
- some of the isolated precursors will constitutively present antigens or have taken up such antigen in vivo in the issue of interest that are targets or potential targets of an immune response for which tolerisation is desired.
- the delivery of exogenous antigen is not essential.
- cells may be derived from biopsies of healthy or diseased tissues, lysed or rendered apoptotic and the pulsed onto dendritic cells.
- the antigen-specific antigen-presenting cells of the invention may be obtained or prepared to contain and/or express one or more antigens by any number of means, such that the antigen(s) or processed form(s) thereof, is (are) presented by those cells for potential modulation of other immune cells, including T lymphocytes and B lymphocytes, and particularly for producing T lymphocytes and B lymphocytes that exhibit anergy to a specified antigen or group of antigens.
- the subject antigen-specific antigen-presenting cells are useful for producing T lymphocytes that exhibit tolerance/anergy to an antigen or group of antigens.
- the efficiency of inducing lymphocytes, especially T lymphocytes, to exhibit anergy for a specified antigen can be determined by assaying immune responses to that antigen including, but not limited to, assaying T lymphocyte cytolytic activity in vitro using for example the antigen- specific antigen-presenting cells as targets of antigen-specific cytolytic T lymphocytes (CTL); assaying antigen-specific T lymphocyte proliferation (see, e.g., Vollenweider and Groseurth, 1992, J. Immunol. Meth.
- CTL antigen-specific cytolytic T lymphocytes
- 149: 133-135) measuring B cell response to the antigen using, for example, ELISPOT assays, and ELISA assays; interrogating cytokine profiles; or measuring delayed-type hypersensitivity (DTH) responses by test of skin reactivity to a specified antigen (see, e.g., Chang et al. (1993, Cancer Res. 53: 1043-1050).
- DTH delayed-type hypersensitivity
- the anergy-inducing antigen-presenting cells of the present invention have the capacity to efficiently present an antigen, or processed form thereof, on one or both of MHC class I molecules and MHC class II molecules.
- the dendritic cells of the invention are capable of presenting antigen, or processed form thereof, on both of MHC class I and class II molecules.
- antigens are acquired by dendritic cells through the exogenous pathway by phagocytosis and have the ability to process exogenous antigen for MHC class I and MHC class II presentation.
- both CD4 + T helper lymphocytes and CTL may be rendered anergic by the antigen-presenting dendritic cells of the invention, and relative proportions can be altered by altering the form in which antigen is provided to the antigen-presenting cell, and the nature of the antigen-presenting cell as discussed above.
- the dendritic cells of the invention can be charged with multiple antigens on multiple MHCs to yield polyclonal or oligoclonal anergy of T lymphocytes.
- the present invention also provides antigen-specific anergic B or T lymphocytes, especially T lymphocytes, which fail to respond in an antigen-specific fashion to representation of the antigen. Moreover, the T lymphocytes actively regulate prior immune responses or subsequent priming to that antigen. The regulation appears to be long lived and is maintained, for example, for at least about 3 months, and preferably years.
- antigen-specific anergic T lymphocytes are produced by contacting an antigen-specific antigen-presenting cell as defined above with a population of T lymphocytes, which may be obtained from any suitable source such as spleen or tonsil/lymph nodes but is preferably obtained from peripheral blood.
- the T lymphocytes can be used as crude preparations or as partially purified or substantially purified preparations, which are suitably obtained using standard techniques as, for example, described in "Immunochemical Techniques, Part G: Separation and Characterization of Lymphoid Cells" (Meth. in Enzymol. 108, Edited by Di Sabato et al, 1984, Academic Press).
- T lymphocytes This includes rosetting with sheep red blood cells, passage across columns of nylon wool or plastic adherence to deplete adherent cells, immunomagnetic or flow cytometric selection using appropriate monoclonal antibodies as described (Cavanagh et al, 1998; Thomas et al, 1993a).
- the preparation of T lymphocytes is contacted with the antigen-specific antigen- presenting cells of the invention for an adequate period of time for inducing anergy in the T lymphocytes to the antigen or antigens presented by those antigen-presenting cells. This period will preferably be at least about 1 day, and up to about 5 days.
- the proliferation of anergic T lymphocytes produced after this procedure is short-lived and they produce IL-10 in an antigen- specific manner.
- a population of antigen-presenting cell precursors is cultured in the presence of a heterogeneous population of T lymphocytes, which is suitably obtained from peripheral blood, together with an NF- ⁇ B inhibitor and an antigen to which a modified immune response is required, or with a polynucleotide from which the antigen is expressible.
- These cells are cultured for a period of time and under conditions sufficient for: (1) the precursors to differentiate into antigen-presenting cells; (2) the level and/or functional activity of NF- ⁇ B in those antigen-presenting cells to be abrogated or otherwise reduced; (3) the antigen, or processed form thereof, to be presented by the antigen-presenting cells; and (4) the antigen- presenting cells to induce a subpopulation of the T lymphocytes to exhibit anergy to the antigen, wherein the subpopulation is characterised by not proliferating and by producing IL-10. This can occur using Ficoll-purified PBMC plus antigen plus NF- ⁇ B inhibitor since such a preparation contains both dendritic cell precursors and T lymphocytes.
- the antigen-specific anergy induced by the antigen-specific antigen presenting cells of the invention involves a mechanism which is distinguishable from certain other forms of non- responsiveness.
- the antigen-specific antigen-presenting cells induce one or more types of antigen-specific regulatory lymphocytes, especially regulatory T lymphocytes.
- regulatory T lymphocytes especially regulatory T lymphocytes.
- Several populations of regulatory T lymphocytes are known to inhibit the response of other (effector) lymphocytes in an antigen-specific manner including, for example, Trl lymphocytes, Th3 lymphocytes, Th2 lymphocytes, CD8 + CD28 " regulatory T lymphocytes, natural killer (NK) T lymphocytes and ⁇ T lymphocytes.
- Trl lymphocytes can emerge after several rounds of stimulation of human blood T cells by allogeneic monocytes in the presence of IL-10. This subpopulation secretes high levels of IL-10 and moderate levels of TGF ⁇ but little IL-4 or IFN ⁇ (Groux et al, 1997, Nature 389:737-742).
- the Th3 regulatory subpopulation refers to a specific subset induced following antigen delivery via the oral (or other mucosal) route. They produce predominantly TGF ⁇ , and only low levels of IL-10, IL-4 or IFN ⁇ , and provide specific help for IgA production (Weiner et al, 2001, Microbes Infect 3:947-954). They are able to suppress both Thl and Th2-type effector T cells.
- Th2 lymphocytes produce high levels of IL-4, IL-5 and IL-10 but low IFN ⁇ and TGF ⁇ . Th2 lymphocytes are generated in response to a relative abundance of IL-4 and lack of IL-12 in the environment at the time of presentation of their cognate peptide ligands (O'Garra and Arai, 2000, Trends Cell Biol 10:542-550). T lymphocyte signalling by CD86 may also be important for generation of Th2 cells (Lenschow et al, 1996, Immunity 5:285-293; Xu et al, 1997, J Immunol 159:4217-4226).
- CD8 + CD28 " regulatory or "suppressor" subset of T lymphocytes can been induced by repetitive antigenic stimulation in vitro. They are MHC class I-restricted, and suppress CD4 + T cell responses.
- NK T lymphocytes which express the NK cell marker, CD161, and whose TCR are V ⁇ 24J ⁇ Q in human and V ⁇ l4J ⁇ 281 in mouse, are activated specifically by the non-polymorphic CD Id molecule through presentation of a glycolipid antigen (Kawano et al, 1997, Science 278:1626-1629). They have been shown to be immunoregulatory in a number of experimental systems. They are reduced in number in several autoimmune models before disease onset, and can reduce incidence of disease upon passive transfer to non-obese diabetic (NOD) mice.
- NOD non-obese diabetic
- ⁇ T lymphocytes have been implicated in the downregulation of immune responses in various inflammatory diseases and in the suppression of inflammation associated with induction of mucosal tolerance.
- the tolerance induced by mucosal antigen was transferable to untreated recipient mice by small numbers of ⁇ T cells (McMenamin et al, 1995, J Immunol 154:4390- 4394; McMenamin et al, 1994, Science 265:1869-1871).
- mucosal tolerance induction was blocked by the administration of the GL3 antibody that blocks ⁇ T cell function (Ke et al, 1997, J Immunol 158:3610-3618).
- the present invention provides means to generate large quantities of antigen-specific lymphocytes by stimulating lymphocytes with antigen-specific antigen-presenting cells of the invention e.g., for minimally at least about 3 days, preferably at least about 5 days.
- the antigen-specific anergy induced by the antigen-presenting cells of the present invention reflects the inability of the antigen-specific lymphocytes to respond to subsequent restimulation with the specific antigen.
- these antigen-specific lymphocytes are also preferably characterised by production of IL-10 in an antigen-specific manner.
- IL-10 is a cytokine with potent immunosuppressive properties. IL-10 inhibits antigen-specific T lymphocyte proliferation at different levels.
- IL-10 inhibits the antigen- presenting and accessory cell function of professional antigen-presenting cells such as monocytes, dendritic cells and Langerhans cells by downregulation of the expression of MHC class II molecules and of the adhesion and co-stimulatory molecules ICAM-1 and B7.1 and B7.2 (reviewed in Interleukin 10, de Vries and de Waal Malefyt, eds., Austin Tex., 1995).
- IL-10 also inhibits IL-12 production by these cells.
- IL-12 promotes T lymphocyte activation and the differentiation of Thl lymphocytes (D'Andrea, et al, 1993, J. Exp. Med. 178:1041-1048; Hsieh et al, 1993, Science 260:547-549).
- IL-10 directly inhibits T lymphocyte proliferation by inhibiting IL-2 gene transcription and IL-2 production by these cells (reviewed in Interleukin 10, de Vries and de Waal Malefyt, eds., Austin Tex., (1995)), and itself promotes antigen- presenting cells that induce regulatory T cells (U.S. Patent No. 6,277,635) (Groux et al, 1996).
- the presence of anergic T lymphocytes may be determined by assaying IL-10 production.
- IL-lOs exhibit several biological activities which could form the basis of assays and units. See, e.g., Coligan (ed) Current Protocols in Immunology (Greene/Wiley, NY, 1989 and periodic supplements).
- IL-lOs have property of inhibiting the synthesis of at least one cytokine in the group consisting of IFN ⁇ , lymphotoxin, IL-2, IL-3, and GM-CSF in a population of T helper cells induced to synthesise one or more of these cytokines by exposure to antigen and antigen presenting cells.
- the antigen-presenting cells are treated so that they are incapable of replication, but that their antigen processing machinery remains functional.
- This is conveniently accomplished by irradiating the antigen-presenting cells, e.g., with about 1500-3000 R (gamma or X-radiation) before mixing with the T cells.
- IL-10 is assayed by ELISA in cell supematants, or by flow cytometric analysis of intracellular staining (O'Sullivan and Thomas, 2002; Rissoan et al, 1999).
- cytokine inhibition may be assayed in primary or, preferably, secondary mixed lymphocyte reactions (MLR), in which case syngeneic antigen-presenting cells need not be used.
- MLRs are well known in the art, e.g., Bradley, pgs. 162-166, in Mishell, et al, eds. Selected Methods in Cellular Immunology (Freeman, San Francisco, 1980); and Battisto et al. (1987, Meth. in Enzymol. 150:83-91, Academic Press). Briefly, two populations of allogeneic lymphoid cells are mixed, one of the populations having been treated prior to mixing to prevent proliferation, e.g., by irradiation.
- the cell populations are prepared at a concentration of about 2xl0 6 cells/mL in supplemented medium, e.g., RPMI 1640 with 10% foetal calf serum.
- supplemented medium e.g., RPMI 1640 with 10% foetal calf serum.
- both controls and test cultures mix 0.5 mL of each population for the assay.
- the cells remaining after 7 days in the primary MLR are re-stimulated by freshly prepared, irradiated stimulator cells.
- the sample suspected of containing IL-10 may be added to the test cultures at the time of mixing, and both controls and test cultures may be assayed for cytokine production from 1 to 3 days after mixing.
- the anergy provided herein involves either a much lowered proliferative responsiveness to antigen, e.g., less than about 50% response, usually less than about 40% response, more usually less than about 5-10% response or less, as compared to non-anergic cells.
- these anergic cells When stimulated with specific antigen, these anergic cells produce less than about 50% and more usually 5-10% or less interferon- ⁇ than non-anergic T lymphocytes in response to antigen. In contrast, they produce no interleukin-4, but 50% more or greater, and more usually 2-5 fold more interleukin- 10 than non- anergic T lymphocytes in response to antigen.
- the antigen-specific antigen-presenting cells described in Section 3 and the anergic lymphocytes described in Section 4 can be administered to a patient, either by themselves or in combination, for modifying an immune response, especially for inducing a tolerogenic response including the induction of an anergic response, and the suppression of a future or existing immune response, to one or more cognate antigens.
- These cell based compositions are useful, therefore, for treating or preventing an unwanted immune response including, for example, transplant rejection, graft versus host disease, allergies, parasitic diseases, inflammatory diseases and autoimmune diseases.
- transplant rejection which can be treated or prevented in accordance with the present invention, include rejections associated with transplantations bone marrow and of organs such as heart, liver, pancreas, kidney, lung, eye, skin etc.
- allergies include asthma, hayfever, food allergies, animal allergies, atopic dermatitis, rhinitis, allergies to insects, fish, latex allergies etc.
- Autoimmune diseases that can be treated or prevented by the present invention include, for example, psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff- man syndrome, thyroiditis, Sydenham chorea, rheumatoid arthritis, diabetes and multiple sclerosis.
- inflammatory disease include Crohn's disease, colitis, chronic inflammatory eye diseases, chronic inflammatory lung diseases and chronic inflammatory liver diseases.
- the cells of the invention can be introduced into a patient by any means (e.g., injection), which produces the desired modified immune response to an antigen or group of antigens.
- the cells may be derived from the patient (i.e., autologous cells) or from an individual or individuals who are MHC matched or mismatched (i.e., allogeneic) with the patient.
- autologous cells are injected back into the patient from whom the source cells were obtained.
- the injection site may be subcutaneous, intraperitoneal, intramuscular, intradermal, or intravenous.
- the cells may be administered to a patient already suffering from the unwanted immune response or who is predisposed to the unwanted immune response in sufficient number to prevent or at least partially arrest the development, or to reduce or eliminate the onset of, that response.
- the number of cells injected into the patient in need of the treatment or prophylaxis may vary depending on inter alia, the antigen or antigens and size of the individual. This number may range for example between about 10 3 and 10 ⁇ , and more preferably between about 10 5 and 10 7 cells (e.g., dendritic cells or T lymphocytes). Single or multiple administrations of the cells can be carried out with cell numbers and pattern being selected by the treating physician.
- the cells should be administered in a pharmaceutically acceptable carrier, which is non-toxic to the cells and the individual.
- Such carrier may be the growth medium in which the cells were grown, or any suitable buffering medium such as phosphate buffered saline.
- the cells may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D-penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNF ⁇ or interleukin- 1 inhibitors, and/or other forms of specific immunotherapy.
- other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D-penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNF ⁇ or interleukin- 1 inhibitors, and/or other forms of specific immunotherapy.
- BAY has been shown to block TNF- ⁇ -stimulated NF- ⁇ B translocation through inhibition of I ⁇ B ⁇ phosphorylation (Pierce et al, 1997).
- Murine BM precursors were incubated for 6 days with GM-CSF and IL-4 to produce BMDCs, in the presence or absence of BAY. All NF- ⁇ B subunits were demonstrable in BMDCs generated in the absence of BAY, and were present in both nuclear and cytoplasmic extracts ( Figure 2a).
- BMDCs generated in the presence of BAY demonstrated NFKB subunit immunoreactivity in the cytoplasm but not the nucleus ( Figure 2a).
- BMDCs generated in the presence of BAY were similar in phenotype to RelB-deficient DCs, in that they lacked cell surface CD40 expression, and expressed reduced levels of MHC class I and class II ( Figure 2b).
- CD86 was expressed at higher levels in BAY-freated DCs than RelB-deficient DCs.
- BMDC populations generated in the presence or absence of BAY had a dendritic morphology, and expressed CDllb and low levels of F4/80, but no CD8 ⁇ , CD3, CD19, Ly6-c, or CD45R (data not shown).
- BAY-freated DCs were administered to animals 7 days before or 7 days after immunisation with mBSA in CFA.
- mBSA-specific immunity was tested 5 d after DC or mBSA administration.
- mBSA-pulsed BAY-freated DCs prevented priming and conferred suppression of mBSA-specific immunity when compared to mBSA-pulsed DCs that had not been exposed to BAY ( Figure 3).
- mBSA-specific T cell proliferation, Ab production and DTH responses were each suppressed after administration of antigen-exposed BAY-freated DCs. The data indicate that DCs in which RelB function is inhibited lack CD40, prevent subsequent priming of immunity, and suppress a previously primed immune response in vivo.
- the lack of suppression of the KLH DTH response also excludes carry over of non-specific suppressive effects by residual soluble inhibitor to draining LN lymphocytes.
- KLH-pulsed BAY-treated wild type DCs conferred suppression of KLH-specific immunity when compared to KLH-pulsed BAY-treated MHC class II " ' " DCs or KLH-pulsed wild-type DCs that had not been exposed to BAY.
- KLH-specific T cell proliferation not shown
- DTH responses were each suppressed after administration of antigen-exposed BAY-treated MHC class IT' " DCs ( Figure 3e).
- the data indicate that MHC class II expression by the injected DCs is necessary for subsequent suppression, thereby providing evidence that injected BAY-treated antigen-exposed DCs are not cross-presented by recipient DCs.
- BMDCs were generated in serum-containing medium either in GM-CSF and IL-4 (control DCs), in GM-CSF alone ("immature DCs"), (Lutz et al, 2000) or in BAY, GM-CSF and IL-4, then pulsed with KLH and injected s.c.
- CD40 deficiency is sufficient to confer suppression of immunity by DCs. Since RelB deficient and BAY-treated BMDCs lacked cell surface CD40, and suppression of immunity by DCs correlated with CD40 expression, we determined whether lack of CD40 expression by antigen-exposed BMDCs was sufficient for suppression of previously primed immunity.
- DCs generated from CD40 " ' " BM in the presence or absence of BAY were similar in phenotype to RelB-deficient DCs, except for higher cell surface CD86 expression ( Figure 5a). DCs generated from BM of H-2 d CD40 " ' " or CD40 + + mice in the presence or absence of BAY were pulsed with KLH and administered s.c.
- cytokine production by CD4 + T cells in LN draining the site of antigen-exposed DC immunisation was compared ex vivo.
- a greater proportion of CD4 + T cells in LN draining the site of injection of either antigen-pulsed RelB " ' " DCs , BAY-treated DCs or CD40 " ' " DCs produced IL- 10 in response to the mitogen PMA (Table 1).
- a greater proportion of T cells primed by KLH- pulsed BAY DCs also produced IL-10 in response to KLH but not to ovalbumin in vitro.
- CD4 + T cells were magnetically sorted by negative selection from recipient spleens 7 days later and 2.5 x 10 5 cells were transferred to wild type mice primed 7 days previously with KLH in CFA.
- Adoptive transfer of CD4 + T cells derived from IL-10 + ' + mice previously treated with KLH-pulsed BAY-treated BMDCs strongly suppressed the subsequent KLH-specific DTH and T cell proliferative responses (not shown) in recipient mice, when compared with CD4 + derived from IL-10 " ' " mice treated with KLH-pulsed BAY treated BMDCs ( Figure 6c).
- the data indicate that antigen-exposed DCs in which RelB function is inhibited induce a population of antigen-specific CD4 + regulatory T cells that regulate immune responses in an IL-10-dependent manner.
- Human PB monocytes were purified from PBMC by immunomagnetic selection using anti-CD14. They were incubated for 48 h in the presence of 800 U/mL each GM-CSF and IL-4 in
- T cells proliferated in response to either immature or mature DC.
- Figure 12 demonstrates that this lack of proliferation was not due to T cell death as the viability of T cells at the end of a 6 day co-culture with BAY-modified or unmodified DC was not reduced. Viability was assessed by flow cytometry using propidium iodide staining.
- DC were differentiated from monocytes in the presence of varying concentrations of BAY, then their expression of CD40 was analysed by flow cytometry, and the DC were used as stimulators in allogeneic MLR.
- the T cell proliferative response was closely correlated with the % DC that express cell surface CD40.
- the data indicate that following incubation with BAY-modified DC, T cells fail to proliferate and to secrete detectable IFN- ⁇ . This effect on T cells appears to relate to the level of CD40 expressed by the DC.
- BMDC Preparation Ficoll-Paque Method using Complete RPMI-1640 The following protocol is used to prepare high quality BMDC:
- the final concentration for mouse BMDC cultures may range between 2 and 15 ⁇ M. Since tolerance induction correlates precisely with CD40 expression by the DC, varying concentrations of BAY are added to BM DC cultures. At day 6-8, yield is check and viability test using PI and CD40 expression by FACS. The minimum concentration that completely suppresses CD40 expression is chosen. Higher concentrations usually are more toxic and affect yield and viability.
- CD40 expression by murine BMDC generated using this protocol is relatively low.
- the present inventors have found that CD40 expression is best quantitated using a biotinylated primary (e.g., from Pharmingen) followed by sfreptavidin-fluorochrome conjugate.
- Certain directly labelled anti-CD40 mAb barely detect CD40 expression by murine BMDC (e.g., from Santa Cruz). If difficulty is experienced in this regard, the BMDC and BAY-freated BMDC can be stimulated overnight with 100 ng/ml LPS. Only BMDC will upregulate CD40 expression.
- Viable DC that lack CD40 expression reliably induce tolerance. This can be tested by s.c. injection of 0.5 x 10 6 OVA or KLH pulsed BAY DC, priming with OVA in CFA 7 days later, and checking OVA-specific DTH 5-7 days later.
- mice were then treated either with a second subcutaneous injection of 5 x 10 5 mBSA-exposed BAY DC or confrol immunoglobulin.
- the data show that suppression of arthritis was equivalent when comparing mBSA-exposed BAY DC, anti-TNF ⁇ or a combination of DC and anti-TNF ⁇ (see Figure 16).
- arthritis could be resuppressed by mBSA-exposed BAY DC after a flare.
- BAY DC administered to arthritic mice alters the isotype of antigen-specific antibodies.
- Sera obtained from mice in the preceding experiment were assessed for mBSA-specific antibodies by ELISA, and the isotype of the antibody was determined using isotype-specific detection reagents. Shown in Figure 17 are the relative quantities of anti-mBSA AB of each isotype from control untreated mice or mice treated with mBSA-exposed BAY DC, followed by IL-l ⁇ flare, then retreated with mBSA-exposed BAY DC.
- mice primed with antigen KLH in complete Freund's adjuvant were primed with mice primed with antigen KLH in complete Freund's adjuvant. Seven days later, groups of 5 mice were freated with either 5 x 10 5 bone marrow derived DC generated in the presence (Bay DC) or absence of BAYl 1-
- mice were tested for skin test reactivity (DTH) to KLH. Shown in Figure 18 is the increment in ear swelling in response to KLH administered intraderrnally at each time point. There is no statistical difference in scores at any time point for mice administered Bay DC + KLH; p ⁇ 0.01 comparing Bay DC with all other primed groups at 1 month time point. Therefore, suppression of antigen-specific responses remains for at least 8 months after a single adminisfration of antigen-exposed Bay DC.
- DTH skin test reactivity
- Dendritic cells Prevention of type I diabetes in NOD mice by NF- ⁇ B " dendritic cells.
- the non-obese diabetes mouse model of spontaneous type I diabetes mellitus was used to assess the capacity of Bay DC to prevent autoimmune disease.
- Four-week old female NOD mice were freated once with 5 x 10 5 dendritic cells, administered subcutaneously.
- Dendritic cells were generated either from syngeneic NOD bone marrow, or from bone marrow cells from NOD mice transgenic for the autoantigen proinsulin, driven by the invariant chain promoter, each in the presence or absence of BAYl 1-7082.
- Onset of diabetes commences around 85 days, with 80-90% diabetic within 200 days.
- Treatment of mice with Bay DC generated from either NOD or proinsulin-NOD bone marrow cells reduces the rate of onset of diabetes at 135 days, when compared with untreated, saline-treated or dendritic cell treated mice ( Figure 21).
- the phenotype ofNF- ⁇ B ⁇ dendritic cells is reproducible with different NF-K.B inhibitors.
- the present inventors examined whether the phenotype of the described dendritic cells that are capable of inducing tolerance when transferred in vivo, is reproducible when dendritic cells are generated with various NF- ⁇ B " inhibitors.
- Dendritic cells were generated from human monocytes for 48 hours in the presence of GM-CSF, IL-4 and in the presence or absence of either 10 ⁇ M of the I ⁇ B phosphorylation inhibitor BAYl 1-7082 or 2.5 or 10 ⁇ M of the proteasome inhibitor PSI (N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala-leucinal).
- the phenotype of dendritic cells generated in the presence of either inhibitor was indistinguishable, in that CD40 expression was suppressed, HLA-DR was reduced and CD86 and MHC class I expression were not changed, relative to immature DC generated in the absence of inhibitors.
- dendritic cells were the only antigen presenting cells capable of inducing antigen-specific tolerance when NF- ⁇ B activation was blocked using BAYl 1-7082, antigen presenting function of bone marrow derived dendritic cells and macrophages was compared in vivo.
- macrophages bone marrow cells were depleted of red cells by ficoll gradient centrifugation then cultured with lxlO 4 U/ml CSF-1 for 7 days as described by Stacey et al, 1996, J Immunol. 157: 2116-2122.
- Dendritic cells were generated from bone marrow as previously described by Martin et al, 2003, Immunity 18: 155-167.
- Dendritic cells or macrophages were generated from bone marrow precursors in the presence of growth factors and 10 ⁇ M Bayl 1- 7082 then washed and exposed to the antigen KLH.
- Recipient mice were primed with KLH in CFA, then administered 5x10 5 DC or macrophages subcutaneously or no treatment.
- One group was not primed with KLH in CFA.
- KLH-specific delayed type hypersensitivity responses were measured on all groups 4 days after adminisfration of antigen presenting cells.
- the results presented in Figure 23 clearly show that bone marrow macrophages also induce antigen-specific tolerance.
- DCs in which NF- ⁇ B activity was suppressed during development from BM precursors, or CD40 deficient DCs expressed levels of CD86 equivalent to those of mature DCs, and higher than those expressed by immature DCs.
- CD40 levels were lower than those expressed by immature BM DCs, and the expression and function of CD40 and NF- ⁇ B could not be induced by inflammatory signals such as LPS or CD40 ligand.
- vz ' tro-derived immature BM DCs share some characteristics of the BAY-treated BMDCs, including modest induction of tolerance, the current data indicate that deficiency in NF- ⁇ B activity, especially RelB activity, leads to the generation of DCs with a unique phenotype.
- BMDCs generated from RelA, c-Rel and p50 deficient mice were demonstrated (Ouaaz et al, 2002).
- BMDCs generated from RelA/p50 doubly deficient mice were more prone to death, and BMDCs generated from c-Rel/p50 deficient mice showed intact CD40 expression and APC function in MLR (when corrected for viability), but reduced IL-12 production.
- LPS-induced up-regulation of MHC molecules, ICAM-1, CD80 and CD86 was unaffected in c-Rel/p50 deficient mice.
- RelA, RelB and c-Rel - partnering with p50 - each play unique and complementary roles in the process of myeloid DC differentiation (Grumont et al, 2001; Neumann et al, 2000; O'Sullivan and Thomas, 2002; Rescigno et al, 1998).
- RelB/p50 specifically controls functional myeloid DC differentiation and CD40 expression.
- a similar role for RelB and CD40 in determining the consequences of presentation of antigen by B cells has also emerged (Buhlmann et al, 1995; Hollander et al, 1996; O'Sullivan et al, 2000; Pai et al, 2002).
- Induction of suppression was specific for the antigen to which DCs had been exposed. Moreover, this suppression results at least in part from induction of antigen-specific regulatory T cells (Treg), as DC immunisation increased the proportion of CD4 + T cells producing IL-10 in draining LN, and CD4 + splenic T cells from tolerant animals transferred antigen-specific tolerance to primed recipients in an IL-10-dependent manner. Therefore, the DCs induced an active "infectious" process of antigen-specific regulation (Cobbold and Waldmann, 1998). While the exact phenotype of the CD4 + Treg induced in the above Examples is not yet elucidated, they most closely resemble Trl cells.
- Antigen specific Trl cells induced by monocytes in vitro produce IL- 10, and are able to suppress inflammation in colitis and allergic models in an IL-10 dependent manner (Cotfrez et al, 2000; Groux et al, 1997).
- human immature monocyte-derived myeloid DCs also induced CD8 + T regulatory cells in vivo, which produced high levels of IL-10 and low levels of IFN- ⁇ , but no IL-4 (Dhodapkar et al, 2001).
- Treg induced Treg were capable of traffic from draining LN to spleen following s.c. administration of DCs. After adoptive fransfer, the Treg are likely to suppress DTH responses locally in the skin, but also suppress T cell proliferation in LN draining the site of antigen priming in the recipient animals.
- NF- ⁇ B The NF- ⁇ B family of proteins is regulated by I ⁇ B and other inhibitory molecules in the cytosol (Baldwin, 1996).
- Cellular activation leads to I ⁇ B phosphorylation and translocation of active NF- ⁇ B to the nucleus.
- RelB is translocated upon myeloid DC differentiation and it heterodimerizes with p50 in the DC nucleus (Neumann et al, 2000; O'Sullivan and Thomas, 2002;
- RelB and p50 deficient mice exhibit multiple deficits in immune function - in particular, RelB deficient mice lack mature myeloid DCs and the liver and spleen are infiltrated by myeloid cells, including monocytes, granulocytes and progenitor cells (Burkly et al, 1995; Sha et al, 1995; Weih et al, 1995).
- the role of NF- ⁇ B in DC APC function was previously examined in vivo through the use of NF- ⁇ B decoy oligonucleotides (Giannoukakis et al, 2000).
- NF- ⁇ B inhibition was commenced several days after the initiation of the DC cultures and CD40 expression by BMDCs was not affected by the NF- ⁇ B decoy.
- BAY 11-7082 has been shown previously to block NF- ⁇ B nuclear translocation tlirough inhibition of I ⁇ B phosphorylation (O'Sullivan and Thomas, 2002; Pierce et al, 1997). While nuclear translocation of all NF- ⁇ B subunits was inhibited in BMDCs, the specificity of the drug for NF- ⁇ B is unknown.
- the present inventors provide two additional pieces of evidence that the consequences of antigen presentation by myeloid DCs are indeed determined by RelB activity.
- RelB deficient BMDCs conferred similar suppression to BMDCs generated from wild type mice in the presence of the BAY 11-7082 inhibitor. Furthermore, the extent of nuclear RelB DNA binding in DCs was inversely correlated with the induction of suppression by those cells.
- the current data highlight the potential for development of antigen-specific autoimmune immunotherapy using DCs or macrophages treated with inhibitors of NF- ⁇ B, in view of the potent suppression of CD40 expression without the need for genetic manipulation of these antigen- presenting cells, and the profound effect on previously primed immune responses in vivo.
- DCs were cultured in RPMI, supplemented with 10% heat-inactivated fetal calf serum (FCS, CSL, Parkville, Ausfralia), 100 ⁇ g/ml penicillin, 100 ⁇ g/ml sfreptomycin, 10 mM sodium pyruvate, 20 mM HEPES, 2 mM L-glutamine and 50 ⁇ M 2-mercaptoethanol (culture medium, CM).
- DCs were cultured in serum free Excell 620 culture medium (CSL Biosciences) supplemented with 100 ⁇ g/mL penicillin, 100 ⁇ g/mL streptomycin, 10 mM sodium pyruvate, 20 mM HEPES, 2 mM L- glutamine and 50 ⁇ M 2-mercaptoethanol (Excell).
- CSL Biosciences serum free Excell 620 culture medium
- BMDCs Bone marrow derived dendritic cells
- Bone marrow cells were collected from murine long bones, suspended by vigorous pipetting, passed through nylon mesh, and the mononuclear cells separated by ficoll gradient centrifugation. Macrophages, class IT cells and lymphocytes were immunodepleted using appropriate mAb followed by magnetic beads (MACS, Miltenyi Biotec, CA). BM cells were incubated for 6-8 days in CM supplemented with 0.5ng/mL recombinant murine GM-CSF and 0.5ng/mL recombinant murine IL-4 (both from Peprotech, USA) and fresh medium was applied every second day. Resulting preparations routinely contained 80-90% CDllc + cells.
- BMDCs were cultured continuously in the presence of 5 ⁇ M BAY 11-7082 (BioMol, Plymouth Meeting, PA) and washed three times before use.
- BAY 11-7082 BioMol, Plymouth Meeting, PA
- BMDCs were incubated for 8 d in CM, supplemented with 0.5ng/ml recombinant murine GM-CSF (Peprotech). Flow Cytometry
- BMDCs were incubated for 30 min on ice with anti-CD86-FITC (GL1, PharMingen, San Diego, CA), anti-CDllc-PE (HL3, PharMingen), anti-CDllb-PE (Ml/70, PharMingen), F4/80-PE (Serotec, Raleigh, NC), CD8 ⁇ -PE (53-6.7, PharMingen), CD3-PE (17A2, PharMingen), or with anti-CD40 (3/23, PharMingen), anti-CD19 (1D3, American Type Culture Collection, ATCC), anti- Ly6-g and c (Grl, RB6-BC5), anti-CD45R (B220, RA3-6B2, each a gift from G.
- anti-CD86-FITC GL1, PharMingen, San Diego, CA
- anti-CDllc-PE H3, PharMingen
- anti-CDllb-PE Ml/70, PharMingen
- lymphocytes were stimulated in the presence of PMA/Ionomycin and brefeldin A for 18 h, then stained with FITC-CD4 (Pharmingen), followed by 4% paraformaldehyde fixation and permeabilization with saponin (Sigma, MO). Permeabilized cells were stained with allophycocyanin (APC) labeled-anti-IL-10, APC-anti-IL-4, or APC-anti-IFN- ⁇ (Pharmingen) for 30 min on ice and washed twice.
- APC allophycocyanin
- Nuclear and cytoplasmic extracts were prepared as previously described (Pettit et al, 1997) and protein estimations carried out using a Protein Assay kit (Bio-Rad, Hercules, CA). 10 ⁇ g of protein extract were separated by 8% SDS-PAGE.
- membranes were immunoblotted with either anti-RelB (sc-226), anti-p50 (sc-7178), anti-c-Rel, anti-RelA, or anti-p52 antibodies (all from Santa Cruz Biotechnology) followed by sheep anti-rabbit HRP-conjugated Ig (Silenus, Hawthorn, Ausfralia) and then detected by enhanced chemiluminescence (ECL, Life Technologies, MO, USA) according to the manufacturer's instructions (Amersham).
- NF- ⁇ B binding ELISA p50 and RelB DNA binding was detected by ELISA using a Mercury Transfactor p50 Kit
- C57BL/6 and BALB/c mice (Animal Resource Centre, Perth, Ausfralia) were maintained in specific pathogen free (SPF) conditions.
- the RelB mutant C57BL/6 mice were originally generated in D.Lo's laboratory (Burkly et al, 1995). They were bred under SPF conditions in the animal facility of the Walter and Eliza Hall Institute (W ⁇ HI). Homozygous RelB " ' " mice were selected and supplied by Dr L. Wu (WEHI) at 5-7 weeks of age for BMDC generation.
- CD40 deficient mice (Kawabe et al, 199 ) were crossed for over 10 generations under SPF conditions with BALB/c mice at the animal facility at Australian National University (ANU), and homozygous CD40 " ' " mice were supplied from ANU at 5 weeks of age.
- IL-10 and MHC class II deficient C57B1/6 mice were bred under SPF conditions and supplied from ANU at 6 weeks of age. All s.c. injections were delivered to the tailbase.
- Mice were immunised s.c. with 60 ⁇ g of mBSA or 50 ⁇ g of keyhole limpet hemocyanin (KLH) or 50 ⁇ g of ovalbumin in complete Freunds adjuvant (CFA).
- mice were injected s.c. or i.v., 7 days before or after the immunisation. Serum antigen-specific Ab, draining lymph node (DLN) T-cell proliferative responses and DTH responses were measured.
- DTH responses mice were injected i.d with either 5 ⁇ g of antigen or saline into the ears and ear swelling was measured and scored 24 h later using an engineer's micrometer.
- C57BL/6 mice were injected s.c. with 5xl0 5 KLH-pulsed BMDCs.
- splenocytes were enriched for T cells by transfer to sterile nylon wool columns (Robbins Scientific, Sunnyvale, CA) for 1 h at 37° C .
- CD3 + CD4 + and CD3 + CD4 " cells were sorted using a Moflo flow cytometer after staining with anti-CD3-FITC and anti-CD4-PE (Cytomation, Fort Collins, CO). Purity was approximately 85%.
- CD3 + CD4 + T cells were purified by immunomagnetic depletion with anti-CD8, anti-MHC class II, Grl, B220, and F4/80. 2.5-5xl0 5 of each purified population was injected i.v. into non-irradiated syngeneic recipients, primed 7-9 days previously with either KLH or ovalbumin in CFA. Antigen-specific T cell proliferative responses were measured in DLN after 7 days.
- T-cell proliferation assay a single cell suspension was prepared from the inguinal LNs. 4xl0 5 LN cells/well were incubated in friplicate in the presence or absence of varying concentrations of antigen or 1 ⁇ g Concanavalin A (Con A, Sigma, MO), at 37° C in 5% CO2 for 3 days.
- mBSA methylated BSA
- assays were carried out in serum free Excell 620 culture medium. Cells were pulsed with 1 ⁇ Ci 3 H- thymidine/well for the final 6-8 h, then harvested onto glass fibre filters using an automated cell harvester. Incorporated 3 H-thymidine was counted using a Packard TopCount NXT (Packard, Meriden, CT). Specific 3 H-thymidine incorporation (cpm) was the mean ⁇ SEM of triplicate wells.
- mice were bled from the lateral tail vein and serum prepared. 100 ⁇ L of mBSA or KLH protein, at 10 ⁇ g/mL in 50 mM carbonate buffer (pH 9.6), was coated onto the wells of 96 well microtitre plates (Griener Labortechnik, Kresmuster, Austria). After washing with 0.5% Tween 20/PBS and blocking with 200 ⁇ L 3% BSA fraction V, 100 ⁇ l serum in five-fold dilutions were added to triplicate wells.
- H-2 mice were injected with KLH-pulsed DCs generated from RelB +/+ or RelB " ' " BM in the presence or absence of BAY (experiment 1), or H-2 d mice were injected with KLH-pulsed DCs generated from CD40 +/+ or CD40 " ' " BM in the presence or absence of BAY (experiment 2).
- H-2 b mice were injected with KLH-pulsed DCs generated from BM in the presence or absence of BAY (experiment 3). Confrol mice were injected with DCs alone.
- DLN cells were stimulated with either PMA/Ionomycin (experiments 1, 2), KLH, or medium alone each in the absence of serum (experiment 3) for 18 h in the presence of brefeldin A then stained with CD4-PE and either IL-10-APC or IFN- ⁇ -APC. Mean ⁇ SD % cytokme-expressing CD4 + T cells from groups of 5 mice tested individually are shown.
- Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population, Nature 362, 761-763.
- TREM-1 amplifies inflammation and is a crucial mediator of septic shock, Nature 410, 1103-1107.
- Plasmacytoid Dendritic Cells Natural Interferon- alpha/beta-Producing Cells Accumulate in Cutaneous Lupus Erythematosus Lesions. Am J Pathol 159, 237-243.
- Progenipoietin- 1 a multifunctional agonist of the granulocyte colony- stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells. Exp Hematol 29, 943-951.
- Dendritic cell modulation by lalpha,25 dihydroxyvitamin D3 and its analogs a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo, Proc Natl Acad Sci U S A 98, 6800-6805.
- Interleukin- 10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med 184(1): 19-29.
- a CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis, Nature 389, 737-742.
- Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival, Transplantation 64, 1808-1815.
- CD40-CD40 ligand interactions augment survival of normal mice, but not CD40 ligand knockout mice, challenged orally with
- Dendritic cells specialised and regulated antigen processing machines, Cell 106, 255-258.
- Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. J Exp Med 178, 1067-1078.
- a conditioned dendritic cell can be a temporal bridge between a CD4 + T-helper and T-killer cell, Nature 393, 474-478.
- TGF-beta 1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors. J Immunol 157, 1499-1507.
- CD40-CD40 ligand interaction is central to cell-mediated immunity against Toxoplasma gondii: patients with hyper IgM syndrome have a defective type 1 immune response that can be restored by soluble CD40 ligand trimer, J Immunol 162, 6690-6700.
- Rheumatoid synovial T cells contribute to the pathogenesis of RA: comment on the article by Matthews et al. (letter).
- Nuclear RelB+ cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions. Immunol Cell Biol 80, 164-169.
- CMRF-44+ monocyte-derived dendritic cells insights into phenotype and function. Exp Hematol 26, 1255-1264.
- CD40 ligand is not essential for induction of type 1 cytokine responses or protective immunity after primary or secondary infection with histoplasma capsulatum, J Exp Med 757, 1315-1324.
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004101776A2 (en) | 2003-05-14 | 2004-11-25 | University Of Debrecen | Novel uses of ppar modulators and professional apcs manipulated by the same |
WO2006108574A1 (en) * | 2005-04-08 | 2006-10-19 | Tgc Biomics Gmbh | Method for the delivery of exogenous antigens into the mhc class i presentation pathway of cells |
WO2008043157A1 (en) * | 2006-10-12 | 2008-04-17 | The University Of Queensland | Compositions and methods for modulating immune responses |
US7988963B1 (en) * | 1999-10-15 | 2011-08-02 | Baylor Research Institute | Use of allogeneic cell lines to load antigen presenting cells to elicit or eliminate immune responses |
CN101646418B (en) * | 2006-10-12 | 2013-07-17 | 昆士兰大学 | Compositions and methods for modulating immune responses |
US9975944B2 (en) | 2013-03-12 | 2018-05-22 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Synthetic peptides for the treatment of autoimmune diseases |
CN110923206A (en) * | 2018-09-20 | 2020-03-27 | 天津贝罗尼生物科技有限公司 | Method for preparing antigen presenting cell |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9930616D0 (en) * | 1999-12-24 | 2000-02-16 | Mathilda & Terence Kennedy Ins | Activation and inhibition of the immune system |
-
2002
- 2002-12-04 AU AU2002953094A patent/AU2002953094A0/en not_active Abandoned
-
2003
- 2003-08-12 EP EP03783854A patent/EP1546310A4/en not_active Withdrawn
- 2003-08-12 CA CA002494675A patent/CA2494675A1/en not_active Abandoned
- 2003-08-12 WO PCT/AU2003/001021 patent/WO2004015056A2/en not_active Application Discontinuation
Non-Patent Citations (9)
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7988963B1 (en) * | 1999-10-15 | 2011-08-02 | Baylor Research Institute | Use of allogeneic cell lines to load antigen presenting cells to elicit or eliminate immune responses |
WO2004101776A2 (en) | 2003-05-14 | 2004-11-25 | University Of Debrecen | Novel uses of ppar modulators and professional apcs manipulated by the same |
WO2006108574A1 (en) * | 2005-04-08 | 2006-10-19 | Tgc Biomics Gmbh | Method for the delivery of exogenous antigens into the mhc class i presentation pathway of cells |
WO2008043157A1 (en) * | 2006-10-12 | 2008-04-17 | The University Of Queensland | Compositions and methods for modulating immune responses |
JP2010505883A (en) * | 2006-10-12 | 2010-02-25 | ザ ユニバーシティー オブ クイーンズランド | Compositions and methods for modulating immune responses |
CN101646418B (en) * | 2006-10-12 | 2013-07-17 | 昆士兰大学 | Compositions and methods for modulating immune responses |
US9017697B2 (en) | 2006-10-12 | 2015-04-28 | The University Of Queensland | Compositions and methods for modulating immune responses |
US9561272B2 (en) | 2006-10-12 | 2017-02-07 | The University Of Queensland | Compositions and methods for modulating immune responses |
EP3590503A1 (en) * | 2006-10-12 | 2020-01-08 | The University of Queensland | Compositions and methods for modulating immune responses |
US9975944B2 (en) | 2013-03-12 | 2018-05-22 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Synthetic peptides for the treatment of autoimmune diseases |
CN110923206A (en) * | 2018-09-20 | 2020-03-27 | 天津贝罗尼生物科技有限公司 | Method for preparing antigen presenting cell |
Also Published As
Publication number | Publication date |
---|---|
AU2002953094A0 (en) | 2002-12-19 |
EP1546310A4 (en) | 2006-05-17 |
EP1546310A2 (en) | 2005-06-29 |
CA2494675A1 (en) | 2004-02-19 |
WO2004015056A3 (en) | 2004-04-01 |
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