DENDRITIC CELL ASSAY FOR IMMUNOMODULATORY MOLECULES

TECHNICAL FIELD THIS INVENTION relates to immunology. More particularly, (his invention relates to use of dendritic cells to identify or screen immunomodulatory molecules having adjuvant or tolerogenic activity.

BACKGROUND

Human immunodeficiency virus (HIV), tuberculosis (TB) and malaria are examples of diseases against which natural immunity is hard to acquire and for which no effective vaccine exists. The causative pathogens all localize within the cells of the host where they effectively evade the humoral immune response. To control such infections, T cell mediated response that can directly kill infected cells, or indirectly mediate the death of the pathogen is required,

Norte of the currently licensed vaccines are designed to induce cellular responses. In fact, it is surprisingly difficult to initiate such responses to non- replicating antigens, which is a contributing factor to the failure to develop vaccines that induce ceil mediated immunity. Dendritic cells (DCs) are critical mediators of the cellular immune response, and recent discoveries have identified, skin-resident DCs as highly efficient initiators of T cell responses, in this context, it is appropriate t recognize that skin-resident DCs are the target of skin- delivered vaccines.

SUMMARY

In work leading to the present invention, cholera toxin was discovered as having a unique and potent capacity to activate skin-resident DCs and induce their migration t the draining lymph node (DL ) where they activate T cells to co- injected antigens. In this context, cholera toxin displayed adjuvant properties towards DCs. Subsequently, an in vitro assay has been developed to rapidly identify compounds with the ability to modulate DC function and induce the properties of an adjuvant capable of activating DCs.

Unexpectedly, the assay can also identify compounds with an inhibitory effect on DCs and may also be useful for identifying novel compounds that can induce immune tolerance and are useful in therapies for auto-immune and other immunological disorders that are responsive to induction of immune tolerance.

In a first aspect, the invention provides a method for determining the immunomodulatory activity of a candidate molecule, said method including the step of contacting an immature dendritic cell with one or more candidate immunomodulators to thereb determine whethe the one or more candidate immunomodulatorshas one or more immunostimulatory properties or immunosuppress i ye properties.

In a particular embodiment, the one or more candidate immunomodulators are adjuvants or have adj vant properties.

In a particular embodiment, the one or more candidate immunomodulators are toleragens or have toleragenic properties.

Suitably, the immature dendritic cell may be a cell line such as DC2.4 or MUTZ3, although without limitation thereto.

In an embodiment the method includes the step of determining an expression level of one or more cell surface markers expressed by the immature dendritic cell. Non-limiting examples include HC-I; MHC-II; CD80; CD86 and/or CD70: and/or chemokine receptors such as CCR7 and CCR5, although without limitation thereto.

In another embodiment, the method includes the step of determining an expression level of one or more secretable factors produced by the immature dendritic cell, or by another cell contacted by the dendritic cell, suc as I L -2. IL- 12, iFN-v, 1L-6 and/or TNF~GL although without limitation thereto.

In some embodiments, said another cell may be a Ύ cell. In yet another embodiment, the method includes the step of measuring phagocytosis b the immature dendritic cell.

In still yet another embodiment, the method includes the step of measuring antigen presentation by the immature dendritic cell.

In a. second aspect, the invention provides a method of determining the immunomodulatory activity of a candidate molecule, said method including the steps of: contacting an immature dendritic cell with one or more candidate imtmrnQmodulators to thereby identify one or more immunomodulators having immunostimulatory or immunosuppressive properties according to the first aspecl; and administering the one or more immunomodulators to an animal, wherein the presence of dendritic cells in the animal that are capable of activating T cells indicates that the one or more immunomodulators have immunostimulatory properties; and the presence of dendritic cells i the animal that are capable of suppressing, inhibiting or tolerizing T cells indicates that, the one or more immunomodulators has immunosuppressive properties.

In a particular embodiment, the method of the second aspect includes the steps of; administering the one or more immunomodulators to a animal in combination with an immunogen; and measuring the immune response to the immunogen to thereby determine whether the one or more candidate immunomodulators has immunostimulatory properdes or immunosuppressive properties.

An embodiment of the aforementioned aspects includes the step of selecting or obtaining the candidate immunomodulator for use as a imm un osti mulant or im.munosupp.res sant.

In a further aspect, the invention provides an immunomodulator molecule identified or identifiable accordin to the method of either o both of the aforementioned aspects.

In one embodiment, the immunomodulator is an immunostimulant. In a particular embodiment, the immunostimulant is an adjuvant. In another embodiment, the immunomoduiator i ^' san immunosuppressant. In a particular embodiment, the immunosuppressant is a toleragen.

In a still further aspect, the inventio provides a method of modulating an immune response including the step of administering the immunomoduiator of the aforementioned aspect to a mamal to thereby modulate an immune response in the mammal.

In one embodiment, the immunomoduiator is administered in combination with an immunogen.

BRIEF DESCR IPTION OF THE FIGURES

In order that the present invention may be more readily understood and placed into practical effect, preferred embodiments of the invention will be described, by way of example only, with reference to the accompanying figures. Figure I, Cells from the mouse DC cell line DC2.4 were cultured overnight with candidate adjuvants (Compound A = Cholera toxin A + B; or Compound G = conifer extract) or media (Media C ) and fluorescent beads. Following overnight culture the cells were harvested and stained with fluorescently labelled antibodies to surface markers of interest and their expression levels determined by flow cytometry ((A) MHC-I expression shown, the numbers in the histograms indicate the MFI). The percentage of DCs that had phagocytosed beads was also assessed durin the flow cytometric analysis (B); and confirmed, by analysis with fluorescent microscopy.

Figure 2. Cells from tire mouse DC cell line DC2.4 were cultured overnight with candidate adjuvants (Compound A = Cholera toxin A + B; or Compound G = conifer extract) or media (Media Ctrl). Following overnight culture the cells were harvested and stained wit fluorescently labelled antibodies to MHC-I, MHC-II and CD86 (A) the numbers in the histograms indicate the MFI. The MFI fo each parameter is shown as a bar graph (B), or normalised to the Media Ctrl (sample MF /Media C MFI) and expressed relative to the Media Ctrl (C) givin an indication of the ability of the test compound to up-regulate, or down-regulate the expression or acti vity of interest,

Figure 3. Cells from the mouse DC cell line DC2.4 were cultured overnight with candidate adjuvants as shown, or media (Media C ). Followin overnight, culture the cells were harvested and stained with fiuorescentiy labelled antibodies to surface antigens as shown. The MFI for each parameter is normalised to the Media Control (sample MFI/Media Ctrl MFI) and expressed relative to the Media Control (A). The results are added (B) and ranked (C) giving an indication of the relative- ability of the test compound to affect DC pheno.type.

Figure 4. Ceils from t e mouse DC cell line DC2.4 or primary bone-marrow derived DC (BMDC) were cultured overnight with candidate adjuvants (as shown) or media, BMDC cultures also contained fluorescent beads. Following overnight culture the ceils were harvested and stained with fiuorescentiy labelled antibodies to surface antigens as show and assessed by flow cytometry. Phagocytosis ('Phago') was assessed as described in Fig 1 (above). The values were normalised to the highest recorded value for each parameter and are displayed as a heatmap (A). The values for all parameters were added (SUM) and the compounds ranked according to their relative activity (B).

Figure 5. Candidate immunostimulants/adjuvants were co-injected with OVA- protein subeutaneously. in the flank of mice and the draining lymph nodes removed after 24hrs, The phenofype of the migrator DC population was determined by flow cytometry (A & B), The putative migratory skin dendritic cell were sorted by flow cytometry and incubated for three days with CFSE labelled OT-I and OT-II (CD8+ and CD4+ T cells that recognize epitopes from ovalbumin (OVA)). The number of divided (live) cells was determined by flow cytometry (C), In parallel experiments, naive mice were vaccinated once with OVA-peptides and candidate adjuvants then challenged seven days later with s.c. injection of OVA-expressing B _. 16 melanoma tumour ceils. Mice were monitored for 80 days and euthanized when tumor size exceeded 1cm ³ (10 mice/group). Figure 6. Calculation method for determining the activity of a candidate immunomodulator. Activity may be either a down-regulation, or up-regulation of expression or activity of the dendritic cell parameter being examined.

Figure 7a and Figure 7b. Overview of an embodiment of the method of the first and second aspects of the invention.

Figure 8. PPC down-regulates p2m gene expression. DC2.4 cells were cultured overnight in the conditions indicated. Cells were harvested, counted and RNA extraction was performed on a known number of live cells. cDNA was prepared and quantified by real-time PCR using primers and probes for β2πι.

Figure 9. Schematic of method for rapidly testing the ability of compounds to modulate derma DC function in vivo. Compounds are injected s.c. or i.d into the flank of test mice and the DLNs recovered 24 h later. DCs responding to the injection travel to the DLN and can be readily identified as migratory DCs. FACS analysis is performed to quantify various migratory DC subsets and to assess their functional phenotype.

Figure 10. Migratory DC populations in DLN following s.c. injection of PPC or CT. Flow cytometric analysis of C57BL/6 DLN cell populations 24h after s.c. injection of OVA + compound as shown into the flank.

Figure 11. The modulatory effects of PPC and CT on DCs can be observed in the phenotype of migratory DCs in the DLN. Phenotype of migratory DCs recovered from the DLN of C57BL/6 mice 24h after s.c. injection of OVA + compounds as shown. Cells were assessed by flow cytometry according to the gating shown in Figure 9. Data are from 5 mice per group; mean and SE are shown. Groups are compared using one-way AN OVA with Uncorrected Fisher's LSD test. RFI = relative fluorescence intensity; LC = Langerhans cells..

Figure 12. Schematic of method for assessing the ability of DCs to activate T cells following injection of immunomodulators. Compounds are co-injected with antigen s.c or i. d. into the flank of test mice and the DLNs recovered 24 h later. DCs responding to the injection travel to the DLN and can be readily identified and sorted into subpopulations by FACS. The sorted DCs are incubated with CFSE-labeled antigen-specific T cells for 3 days following which they are assessed by FACS for viability, number and proliferation.

Figure 13. In vivo PPC exposure severely inhibits the ability of DCs t activate CD8+ T cells. Migratory DCs recovered from the DLN 24h after s.c, injection of PBS, or PBS + OVA or OVA and CT or PPC were sorted by FACS into CD 103+ or CD103- populations and incubated with naive (MACS-purified and CFSE- labeled) ΟΤ-Γ CD8+ T cells (DC:C)T-I ratio = 1 :10) for 3 days. Following the incubation the cells were harvested and stained with antibodies against CDS and Vot2 and assessed by flow cytometry. (A) Representative FACS plot from one well of each group showing all events. (B) Graphs of cell recovery for each group. Dead cells were excluded on the basis of propidium. iodide uptake. Injection groups included 5 mice/group and DLN were then pooled for each group for sorting. Bars indicate means and SE for replicate wells.

DETAILED DESCRIPTION

The present invention is at least partly predicated on the discovery that immature dendritic cells (DCs) are particularly suitable for determining whether an immunomodulator has inimuuostimulatory (e.g adjuvant) properties or immunosuppressive (e.g. tolerogenic) properties. Furthermore, the discovery that cholera toxin activates skin DCs and induces their apparent migration to the draining lymp node (DLN) where they activate T cells to co-injected antigens makes these cells useful in animal models for further in vivo screening of immunomodulators to determine whether they have immunostimulatory (e.g adjuvant) properties or immunosuppressive (e.g. tolerogenic) properties,. The feci that the aforementioned methods revealed that a pine cone extract (referred to herein as Compound G or PPC) previously thought to be immunostimulatory actually had an inhibitory effect on DCs demonstrates the unexpected usefulness of the aforementioned methods.

Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-slated integers or groups of integers.

It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "an" iramunomodulator includes one immunomodulatory one or more immmiomodulators o a plurality of immunomodulators.

By "protein " is meant an amino acid polymer comprising natural and/or non-natural amino acids, D- or L- amino acids as are well known in the art.

A "peptide " is a protein having no more than fifty (50) amino acids.

A "polypeptide" is a protein having more than fifty (50) amino acids.

As used herein an "antigen ⁵ ' is a molecule which is capable of being recognized, bound or responded to by one or more cellular or molecular elements of the immune system. By way of example, an antigen may be a protein, carbohydrate, lipid, nucleic acid or a molecule compri sing one or more of these An "an tibody ' refers to an i m munoglobulin protei n capable of binding an antigen. Antibodies include monoclonal and polyclonal antibodies, recombinant antibodies, fragments such as Fab and Fab'2 fragments, diabodies and single chain antibod fragnients (e.g. scVs), although without limitation thereto. An antibody may further comprise a label selected from a group including an enzyme, a fluorophore, a chemiluminescent molecule, biotin, a radioisotope or other label. Fluoropfiores may be fluorescein isothiocyatiate (FITC). tetramethylrhodamine isolhioeyanaie (TRITC), allophycocyanin (AFC), Texas Red (T ), TAM A, LC red, HEX, FAM, TET, ROX, Cy dye such as Cy3 or Cy5 or R-Phycoeryt rin (RPE) or derivatives thereof, although without limitation thereto.

By "immunogen" or "immimogenic" is meant a molecule that is capable of eliciting an immune response upon administration to an animal, such as a human, either alone or in combination with one or more other molecules such as an adjuvant or other imnrunostimulatory molecule. By wa of example, an immunogen may be a protein, carbohydrate, lipid, nucleic acid or a molecule comprising one or more of these.

A "nucleic acid" as used herein includes sing!e-or double-stranded mRNA, RM A, cRNA, RNAi, siRNA and DNA inclusive of cDNA, mitochondrial DNA (mtD A) and genomic DNA.

I a first aspect, the invention provides a method of determining the immunomodulatory activity of a candidate molecule, said method including the step of contacting an immature dendritic cell with one or more candidate immunoniodulators to thereby determine whether the one or more candidate -Immunomodulatory has immun stimulatory properties or immunosuppressive properties.

As used herein, an "immature dendritic ceil" is a dendritic cell or cell line that is not mature, in the sense that it is capable of phenotypic and functional changes induced by contact with one or more immunomodulator molecules. Immature dendritic cells may be derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells characterized by high endocytic activity and low T-cell activation potential.

In some non-limiting embodiments, it ma be advantageous to define, or at least partly define, immature dendritic cells, or populations of immature dendritic cells, according to a cell surface marker phenotype. An immature dendritic cell, or population of immature dendritic cells, may be defined by expression, or a lack of expression, of one or a plurality of cell surface markers. These may be measured or defined in absolute terms such as "negative" or "positive" or by relative terms such as "low", "medium" or "high" or sometimes represented as "+/-", "++" or "+++ ^" ". By way of. example only, cell surface markers that may be of assistance for defining immature;- dendritic cells, or populations of immature dendritic ceils, include one or a plurality of FcR, Clq complement receptor, MHC-II, CD24, CD80, CD86, CD40, CD2 _. 5, CD83 IL-12 and/or p55, although without limitation thereto. In one particular non-limiting embodiment, immature dendritic cells express high FcR, high Clq complement receptor, low surface MHC-II, low CD24, CD80, CD86, CD40, CD25. CD83. low IL-12 and/or p55, although without limitation thereto.

Activation of immature dendritic cells results in maturation, which includes migration to draining lymph nodes, cytokine production and upregulation of cell-surface markers, such as CD86, CD80 and CD40, greatly enhancing their ability to acti vate T cells.

Mature dendritic cells include CD 103+ dermal .dendritic cells (dDC), which are a subset of skin dendritic ceils that migrate to lymph nodes and efficiently cross-present antigens to CD8+ T cells, in particular.

The immature dendritic cell may be of human, mouse or othe mammalian origin. The immature dendritic cell may be a primary cell (e.g an immature dendritic cell isolated or obtained from a mammal, such a human) or may be a cell line. Suitably, the immature dendritic cell is an immature dendritic cell line of human or mouse origin, suc a DC2.4 or MUTZ3.

By "immunomodulatof" is meant a molecule that modulates, modifies, inhibits, suppresses, augments, increases or enhances one or more immunological features, acti vi ties or properties of a dendritic cell.

In one form, the immunomodulator may be immunostimulant or have imrnunostimulatory activities or properties. In. some embodiments, the immunostimulant is characterized by augmenting, increasing or enhancing one or more immunological features, activities or properties of a dendritic cell, in one particular embodiment, the immunostimulant may be- an adjuvant or have adjuvant features,, activities or properties such as augmenting, increasing or enhancing one or more immunological features, activities or properties of a dendritic cell, suc as an immature dendritic cell.

In another form, the immunomodulator may be an immunosuppressant or have immunosuppressive activities or properties. In one embodiment, the immunosuppressant or im.munostiraulatory activities or properties may be characterized by inhibiting or suppre ing one or more immunological features, acti vities or properties of a dendritic cell. En one particular embodiment, the immunosuppressant is a toleragen that inhibits, reduces or suppresses one or more immunological features, activities or properties of a dendritic cell, suc as an immature dendritic cell.

As generally used herein, terms such as "augmenting, increasing or enhancing ^" ' and 'Inhibits, reduces or suppresses" in the context of activities or properties (such as cell surface marker expression, factor expression and/or secretion, phagocytosis, T cell activation etc) may be measured, detected and/or expressed in relative or absolute terms, as is well understood in the art. In some embodiments, relative measurements; may be relative to a reference cell, such as: an immature dendritic cell not exposed to the candidate immunomodulator; an immature dendritic cell exposed to an immunostimulant; or an immature dendritic cell exposed to an immunosuppressant, as appropriate,

immunological properties of dendritic cells, inclusive of immature dendritic cells, include expression of cell surface markers such as MHC-I; MHC- Π; CD40; CD80; CD86, CD70 and/or chemok e receptors such as CCR7 and CCR5, production of one or more secretable factors such as IL-2, lL-12, IFN-y, IL-10, IL-6 and/or TNF-a, phagocytosis by the dendritic cell and antigen cross- presentation to T cells, although without limitation thereto.

Accordingly, the one or more immunological features, activities or properties of the dendritic cell may by measured or detected with reference to the dendritic cell or with reference to one or more other cells (e.g. T cells) responsive to changes in dendritic cell features, cell surfrace phenotype, activities or properties.

Typically, the immunomodulator is not a. known dendritic cell growth, maturation, survival ^' and/or differentiation factor such as IL-Ιβ, CD40L, GM- CSF, IL-4, ΓΡΝ-γ, VEGF, TNF-oc, IFN-β or TGF-β 1. or other itnrnunostim latory molecule such as an activator of dendritic cell toll-like receptors (TLRs), although without limitation thereto.

Suitably, the candidate immunomodulator is, or comprises, an exogenous molecule, or mixture of exogenous molecules, having unknown, incorrectly or incompletely ascribed immunomodulatory properties and activities. In some embodiments, the candidate immunomodulator may have an incorrectly or incompletely ascribed immunomodulatory activit such as Compound G in the Examples. This candidate immunomodulator was thought to be- an irnmunosrimulant or adjuvant, but instead displayed immunosuppressive or tolerogenic properties according to the method of the invention.

In certain embodiments, the immunomodulator is, or comprises, an exogenous molecule, or mixture of exogenous molecules that are present in, or obtained from, a natural source. Non-limiting examples include plant extracts and animal extracts.

I certain other embodiments, the immunomodulator is, or comprises, an exogenous molecule, or mixture of exogenous molecules that are present in, or obtained from, a library of synthetic molecules. Non-limiting examples include libraries ¾f synthetic peptides, libraries of small organic molecules, phage display libraries, combinatorial libraries and the like.

In an embodiment, the method includes the step of determining, an expression level of one or more cell surface markers such as MHC-I; MHO II; CD80; CD86 and/or CD70; and/or ehemokine receptors suc as CCR7 and CCR5, although without limitation thereto. Suitably, an increase in expression of one or more of said cell surface markers is indicative of immature dendritic cell activation and/or maturation, such as in response to an immunostimulant.

Suitably, a decrease in expression of one or more of said cell surface markers is indicative of suppression or inhibition of the immature dendritic cell, such as in response to an immunosuppressant

Suitably, the cell surface markers are detected by specific antibodies that bind the cell surface marker. Typically, each antibody is labeled with a label, such as a fliioropbore, directly or indirectly (e.g via a blotm-avidin interaction). A preferred detection method includes flow cytometry to determine an expression level of one or more ceil surface markers.

In one particular form of this embodiment, the migratory potential of the immature dendritic cell is measured by expression of eheraokine receptors suc as CCR7 and CCR5. Suitably, an increase in expression of CCR7 and CCR5 is indicative of immature dendritic cell activation and/or maturation.

In a preferred form, migratory potential is determined by an increase in chemokine receptors such as CCR7 and CCR5.

In another embodiment, the method includes the step of determining a level of expression or secretion of one or more factors produced by the immature dendritic cell such as IL-2, IL-12, IFN-y, IL-6 and/or TNF-a, although without limitation thereto. Suitably, an increase in expression of one or more of said factors i indicative of immature dendritic cell activation and or maturation, such as by an immunostimulant. Suitably, reduced or absent expression of one or more of said factors is indicative of immature dendritic cell suppression or ^'■ inhibition, such as by an immunosuppressant..

Levels of expression and/or secretion of said factors may be measured by detecting or measuring the factor or a nucleic acid encoding the factor in embodiments where the factor is a protein. Detection or measurement may be by way of an immunoassay (e.g. cytokine bead assay, intracellular staining, ELISA or radioimmunoassay) or by cell-based assays (e.g. using factor-dependent ceil lines) that qualitatively or quantitatively detect and/or measure- the factor, or by use of reporter genes, (e.g. green fluorescent protein expressed under the influence of the IL-1.2 promoter) although without limitation thereto.

Preferably, factors are detected or measured by a cytokine bead assay.

In other embodiments, a nucleic acid encoding the facto may be detected and/or measured qualitatively or quantitatively by a nucleic acid detection assay such as using polymerase chain reactio (PCR). PCR may include multiplex PCR to simultaneously amplify and detect nucleic acids encoding a plurality of different factors and/or quantitative methods such as real-time PCR and competitive PCR.

i yet another embodiment, the method includes the step of measuring phagocytosis b the immature dendritic ceil. Suitably, an increase in phagocytosis is indicative of immature dendritic cell activation and/or maturation, such as by an immunostimulant. Suitably, reduced, inhibited or absent phagocytosis is indicative of immature dendritic cell inhgibition or suppression, such as by an immuno suppres san t.

Phagocytosis may be measured by any assay, method or technique known in the art. Generally, phagocytosis is measured by determining the ability of the immature or mature dendrific cell to engulf, take up or digest another cell or cell derivative, particle or molecular complex. Typically, the method includes phagocytosis of a particle such as a bead (e.g. a latex bead) or a cell derivative such as a yeast cell wall extract (e.g. Zymosan). Suitably, the cell or cell derivative, particle or molecular complex is labeled with an enzyme (which enables colorimetrie detection via a substrate), digoxigenin or fluorophore to facilitate detection and measurement of phagocytosis.

Preferably, phagocytosis is detected or measured by fiuorophore-labeled bead uptake by the immature dendritic ceil, or mature dendritic cell derived therefrom. In a further embodiment,, the method includes the step of measuring antigen cross-presentation by the- .immature dendritic cell or a mature dendritic cell derived therefrom. Suitably, an increase or enhancement of antigen cross- presentation indicates immature dendri tic cell acti vation and/or maturation.

In one embodiment, cross -presentation is determined or measured by detection of an antigen-MHC comple expressed or presented by the dendritic cell Suitably, detection of the complex is by way of an antibody which is specific for the antigen-MHC complex. By way of example, immature dendritic cells, or mature dendritic cells derived therefrom, may be contacted with antigen and subsequently an antigen-MHC complex detected by the antibody. Typically, the antigen-MHC complex comprises a "processed" peptide derived from the antigen. A non-limiting example is the ovalbumin-derived SIINFE L peptide, wherein immature dendritic cells, or mature dendritic cells derived merefrom, are exposed to ovalbumin before detecting SIINFEKL-MHC Class I complexes expressed by the dendritic cell.

In other embodiments, antigen cross-presentation is measured or determined by the ability of an immature dendritic cell, or a mature dendritic cell derived therefrom, to present antigen to an antigen-specific T cell in an MHC- restrieied manner. Typically, for CD4+ T ceils antigen cross-presentation is in the context of MHC Class II and for CD8+ T cells antigen cross-presentation is in the context of MHC Class I Suitably, antigen cross-presentation activates antigen-specific T cells, which activation can be measured by any assay known in the art including increased T cell proliferation (e.g. ^J H-tliymidine incorporation, or dilution of CFSE), cytokine production (e.g. IL-2 or IFNy) or target cell lysis by activated cytotoxic lymphocytes (CTL), although without limitation thereto.

It will be appreciated that in some embodiments it may be advantageous to determine a concentration of a candidate immunomodulalor (i.e. from a titration) with the most activity. This activity ma be either a dow -regulation, o up- regulation of the immature dendritic cell property being examined as hereinbefore described.

By way of example, to determine the maximal change in activity and the concentration at which that occurred, data from flow-eytometrk analysis may be manipulated as follows:

1) the MFI (geometric mean fluorescence intensity) from flow cytometric analysis is averaged for each titration of each candidate imrnunomodulator and divided by the average MFI of media -only controls, to thereby provide a relative MFI for each titration ("a"), wherein candidate immunomodulators displaying down-regulation will have 0<a< 1 and those with up-regulation will have 1<α ;

2) a is subtracted from 1 to give β which will have a negative value for samples that were up-regulated and a positive value for down-regulated samples;

3) the absolute value of β i s found (y);

4) the maximum value of γ is determined (δ), this represents the value in the array with the greatest variation from the media Ctrl;

5) the relative position of δ in the array is found (ε), and the maximum change from the control sample is determined (ζ) and finally the concentration at which the maximal response occurred is determined.

in one particular embodiment of the method of the first aspect, a plurality of determinations or measurements that include: an expression level of one or more cell surfaee markers on said immature dendritic cell; an expression level of one or more secretahle factors produced or secreted by the immature dendritic cell; phagocytosis b the immature dendritic cell; and antigen presentation by the immature dendritic cell; are measured or assessed in combination, to thereby determine whether the candidate imrnunomodulator has immunostimulator (e,g adjuvant) or immunosuppressive (e.g tolerogenic) activities or properties.

In some embodiments, a ranked sum may be obtained or calculated from this combinatorial, analysis, whereby a relative value may be given to each of a plurality of candidate immunomodulators. Typically, candidate immunomodulators havin a lower relative value would be possible immunosuppressants (e.g tolerogens) and candidate immunomodulators having a higher relative value would be possible immunostimulants (e.g adjuvants).

A non-limiting example of a ranked sum analysis is provided in the Examples, with particular reference to Fig. 4.

In second aspect, the invention provides a method of determining the immunomodulatory activity of a candidate molecule, said method including the steps of: contacting an immature dendritic cell with one or more candidate immimomodulators to thereby identify one or more immunomodulators having immunostimatory (e.g adjuvant) or immunosuppressive (e.g tolerogenic) properties according to the first aspect as hereinbefore described; and administering the one or more immunomodulators to an animal, wherein the presence of skin dendritic cells in the animal that are capable of activating T cells indicates that the one or more immunomodulators has immunostiinulatory (e.g adjuvant) properties; and the presence of skin dendritic cells in the animal that are capable of toleriang T cells indicates that the one or more immunomodulators has immunosuppressive (e.g tolerogenic) properties.

Suitably, one or more candidate immunomodulators that show activity according to the method of the first aspect, typically performed i vitro, are subsequently assayed or tested in vivo in said animal.

In a particular embodiment, the method includes the steps of; administering the one or more immunomodulators to an animal in combination with an immunogen; and measuring an immune response to the immunogen to thereby determine whether the one or more candidate immimomodulators has immunostimulatory (eg adjuvant) properties or immunosuppressive (e.g tolerogenic propertie s .

In some embodiments, the one or more candidate immunomodulators are injected subcutaneously with the immunogen and dendritic cells are isolated from, the animal. Typically, the cell surface marker phenotype of the dendritic cells is assessed, such as by flo cytometry as hereinbefore described. Suitably, skin dendritic cells are isolated and contacted with CD8+ and/or CD4+ T cells that are capable of responding to tile immunogen. h certain embodiments, the ability of the immunogen and dendritic cells to induce T ceil proliferation is indicative of the immunostimulatory (e,g adjuvant) activity of the one or more candidate i mm unomodul a tors .

In other embodiments, the ability of the one or more candidate immunomodulators to induce antigen-specific CD4 ⁺ and CDS* T cells is assessed by quantifying the number of adoptively transferred, antigen-specific T cells recovered after vaccination with candidate immunomoduiator and immunogen. Further characterization of dendritic ceils and responding; T cells is performed. This may include assessment of cytokine production (e.g. protein and/or or encoding nucleic acid) and/or cytolytic function of CTL.

In one particular embodiment described in more det il in the Examples, an immunusuppressant (such as Compund G or PPC) may suppress or inhibit the ability of dendritic cells to activate CDS* T cells,.

In yet further embodiments, candidate immunomodulators identified as having potential to activate T cell immune responses in vivo are further tested by for protective capacit against tumou challenge.

Suitably, the animal is a non-human mammal, suc as a mouse, rat, rabbit or hamster.

An embodiment of the aforementioned aspects includes the step of selecting or obtaining the candidate immunomoduiator for use as an imm un o sti mulant or imm.unosupp.res sant.

Accordingly, a further aspect of the invention provides an immunomoduiator molecule identified or identifiable according to the method of either or both of the aforementioned aspects.

In one embodiment, the immunomoduiator is an immunostimulant. In a particular embodiment, the immunostimulant is an adjuvant. In another embodiment, the immunomoduiator is an immunosuppressant. In a particular embodiment, the immunosuppressant is a toleragen.

A still further aspect, the invention provides a method of modulating an immune response including the step of administering the immunomoduiator of the aforementioned aspect to a mamal to thereby modulate an immune response in the mammal.

in one embodiment, the immunomoduiator is mi imnumostimulant. In a particular embodiment, the immunostimulant is an adjuvant.

In another embodiment, the immunomoduiator is an immunosuppressant. In a particular embodiment, the immunosuppressant is a toleragen.

I one embodiment, the immunomoduiator is administered in combination with an immunogen. It will be appreciated that the imutiogen may be administered before, together with or after administration of the immunomoduiator.

In some embodiments, the immunomoduiator and optionally, the immunogen, may be administered as a pharmaceutical composition. Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent or excipient. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, pQlyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, sugars, sugar alcohols, organic acids such as acetates, propionates and maionates, and pyragen-free water.

In some particular embodiments, the pharmaceutical composition may further comprise one or more immunomodifiers inclusive of adjuvants and immunostimulatory nucleic acids including but not limited to TLR agonists, lipopolysaccharide and derivatives thereof such as MPL, Freund's complete or incomplete adjuvant, hexadecylamine. octadecylamine, octadecyl amino acid esters, lysolecitb in , dimethyidioetadecylammoni urn bromide, N,N-dicoe.tadecyl- Ν', N' is(2¾ydfo.xyethyl-pfOpatte!diaffline), memoxYhexadeeylglyeerol, and pluronic polyols; poly amines such as pyran, dexti¾mulfate,. poly IC carbopoL peptides such as muramyl dipeptide and derivatives, dimethylglyeine, tuftsin, oil emulsions, mineral gels such as aluminum phosphate, aluminum hydroxide or alum, lymphokines, Imiquimod, Guardiquimod, QuilA and immune stimulating complexes (ISCOMS).

In a preferred aspect, the method of modulating a immune response may be suitable for treating or preventing a disease, disorder or condition in a mammal.

In one embodiment, the disease, disorder or condition is responsive to administration of an immunostimulant. Noil -limi ing examples include diseases or conditions caused by pathogens such as bacteria, protozoa and viruses, although without limitation thereto. Other non-limiting examples include diseases or conditions where the mammal is imm nosupressed, such as through viral infections (e.g HIV, CMV or EBV) or a a result of chemotherapeutic or radiation treatment of cancers.

In an alternative embodiment, the disease, disorder or condition is responsive to administration of a immunosuppressant. Non-limiting examples include diseases or conditions caused by, or associated with, inappropriate, excessive or mis-directed immune activity (e.g against self), such as inflammatory and/or autoimmune diseases or conditions, as are well known in the art. By way of example only, these may include respiratory, immunological, endocrine and gastrointestinal diseases or conditions such as inflammatory bowel disease, Chrohn's disease, asthma, autoimmune diabetes, COPD, multiple sclerosis and Hashimoto's thyroiditis, although without limitation thereto.

Tha mammal may be an human or non-human mammal, inclusive of domestic pets, livestock and performance animals, although without limitation thereto.

In order that the invention may be readily understood and put. into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

Materials & Methods

Prepare bulk cultures of DC cell lines, BMDCs or PMDC according to standard protocols. Harvest cells from culture, wash and plate out into 96-weil flat-bottom tissue culture plates at 50.000 eells/well in 50μ1 of suitable media containin 1 drop/ml of FITC-labelled Ι .Ομηι Flow Check microspheres (Polyseience hie). Prepare (2 concentration) mid-log titrations of test compounds over 4 wells in duplicate and add 50μ1 of titrated compound t the wells containing the cells and media (bringing final volume to ΙΟΟμΓ), mix and incubate overnight at 37°C and 5% CQi.

For assessment of cross-presentation by mouse cells: prepare cells as above but with the addition of ovalbumin at a final concentration 5rog/ml, The MHC of the cells must be compatible with the detection antibody (i.e. presently only an anti-SIINFEKL-H2 ^b antibody is available).

After overnight culture, spin plates (600 CF, 4 rain) and transfer 50μ1 aliquots of supernatant by multichannel pipette to suitable 96 well, plates for freezin (tightly sealed). Add ΙΟΟμ.1 of trypsin buffer (PBS with 30% trypsin and 10% FCS) to the cells and harvest into 96-well V-bottomed plate, spin as above, aspirate and re- suspend in 16μ1 of antibody-stainin mix prepared in FCAB buffer (Apte et al., 2011. Int. J. Parasitol 41 1285). Incubate at 4°C for 10 -minutes then wash with 135μ1 of FCAB buffer. Re-suspend in 35μ1 of FCAB buffer with an appropriate dead-cell detection stain (such as propidium iodide). The plates are run on a suitable cytometer equipped with a high -throughput plate reader (the stopping gate is set to 10,000 events, each plate takes approximately 15 minutes to read).

Post-acquisition data analysis is performed with suitable software that has the capacity to handle a multitude of files and parameters simultaneously (such as Flow Jo), The viability of the samples are assessed by the frequency of dead cells in the sample and further analysis is performed on live cells; the viability can be reported as relative to media only control samples. To assess the relative expression levels of activation or other markers, the geometric mean of the sample's fluorescence intensity (determined by flow cytometry) is divided by the geometric mean of control samples (average of 4 replicates) that were incubated overnight i media only (but with the addition of ovalbumin and beads if relevant). This is described in further detail below.

It is necessary to determine the concentration (from the titration) with the most activity: this activity may be either a down-regulation, or up-regulation of expression/activity of the DC component being examined. To determine the maximal change in activity and the concentration at whieh that oecurred, the dat from the fiow-cytometric analysis is exported to a spreadsheet and calculations are made as follo s in the example in Fig 6 whieh shows results from the testing of one parameter (CD86) wit two different candidate adjuvants (Compound A and Compound G):

1) the MFI of the test samples (geometric mean fluorescence intensity) from the flow cytometric analysis is averaged for eac titration and divided by the average MET of (he media-only control samples, this gives the relative MFI for each titration (shown as "a" in Fig 6), samples displaying down-regulation will have 0<a< 1 and those with up-regulation 1<α ;

2) a is subtracted from 1 to give β which will have a negative valu for samples that were up-regulated and a positive value for down-regulated samples;

3) the absolute value of β is found (γ);

4) the maximum value of γ is determined (δ), this represents the value in the array with the greatest variation from the media Ctrl;

5) the relative position of δ in the array is found (ε), and the maximum change from the control sample is determined (ζ) and finally the concentration at which the maximal response occurred is determined.

Results

In vitro ssay

The ability of a compound to activate DCs and induce functional characteristics necessary for the DCs to initiate T cell responses is tested as follows; DCs derived from cell lines (mouse and human) or primary cells from bone-marrow derived DCs (mouse, BMDC) or peripheral blood mononuclear cell-derived DCs (human, PMDC) are co -cultured for 24 hr with a titration of the compound of interest and fluorescent micro-beads (and for mouse cells, with the model antigen ovalbumin (OVA)); or wit appropriate controls. Cells are then assessed by multi-parameter flow cytometry to determine activation by upregulation of co-reeeptors such as (but not limited to) CD80, CD86, CD70, MHC-T, MHC-II) (as in Pig, I A ), increase in phagocytic potential (by quantification of fluorescent bead uptake. Fig. IB), migratory potential (by increases in ehemokine receptors such as CCR7 and CCR.5), and the ability to induce cross-presentation (by measurement of SIINFE.KL-MHC-I. complexes - with antibody to complex). Culture supematants are collected and frozen to allow fo later assessment of the presence of key cytokines from promising lead compounds (assessed by cytokine bead array (CBA) e.g. IL-2, IL-12, IFN-γ. IL-6, TNF, IL-10)).

The results for the test-compounds are normalised to the results from controls cultured in media only; the concentration of the titrated test compound that gave the maximum variation from the control sample is used as the nominated concentration for further assessment: thi gives a rapid indication of the ability of the test compound to up-regulate, or down-regulate the expression or activity of interest (Fig 2). Compound A - Cholera toxin (complete A + B); Compound C » CpG/Alum; Compound F « ^· Iscom (Ahisco); Compound G ~ conifer (pine cone) extract.

We used the assay to detect the ability of twelve different candidate- adjuvants to activate DCs: mouse cells from the DC cell line DC2.4 were cultured overnight with the compound of interest as described above. The following day the expression level of a range of markers was determined by flow cytometry (Fig 3A). The result were normalised to the media-only control; the maximum variation in the titration determined and the test parameter result expressed as a fold change, A simple analysis was performed by adding the normalised value result for each compound (Fig 3B). The compounds were then ranked according to their score (Fig 3C). We performed a similar experiment with primary mouse BMDCs: the cells were cultured overnight with the candidate adjuvant and fluorescent beads (for determination of phagocytosis). The following day the expression of activation markers and phagocytosis was determined as described above, The results from both assays are presented as a heatmap in Figure 4. These activities result in a rapid and comprehensive snapshot of the potential bioactivity of the product.

In vivo assay

Compounds that show strong activity in vitro are tested in vivo in mice as follows: the compound is injected subcutaneousiy (s.e.) with OVA-protein and the draining lymph nodes (DLN) are recovered after 24 h and the phenotype of the migratory DCs in the LN is assessed by flow cytometry (as in Fi 5A & 5B). The migratory skin DC populations have been exposed to the protein and adjuvant by the vaccination and primed to activate T cells. These cells are sorted from the DLN by FACS and incubated for three days with CFSE labelled OT-I and OT-II (CD8+ and CD4+ T cells that recognize epitopes from OVA). The number of divided (and live) cells is determined by flow cytometry (Fig 5C) and gives direct measure of the ability of the test adjuvant to induce T cell proliferation.

In parallel studies tile ability of the compound to induce the expansion of antigen specific CD4 ⁺ and CD8 ⁺ T cells is assessed by quantifying the number of adoptively transferred ΟΤ-Π and OT-I cells recovered one week after vaccination with the compound of interest and QVA-protein (not shown). Further characterization of FACS sorted DCs and responding T cells is performed including: assessment of cytokine production (CBA and qRT-PCR assessment of cytokine mRNA) and cytolytic function of T cells in cognate CTL assay (not shown).

Compounds identified with potential to activate cellular responses in vivo are further tested for protective capacity in tumour challenge- experiments: mice are vaccinated prophylactic all with the compound of interest and OVA-peptide (or protein) and challenged one week later by s-.c, injection of B16-OVA cells and tumour growth monitored (Fig 5D); alternatively, for therapeutic challenge, mice are challenged first with s.c, injection of B 1.6-0 VA and vaccinated after one week as above and monitored. This comprehensive assessment identifie lead compounds with DC activity and characterises then ^* functional properties in vivo.

Compounds with tolerogenic potential ma be tested in vivo as follows (but without limitation): female mice are vaccinated with syngenic killed male splenocytes (antigen) to induce an immune response to the male minor histoeompatability HY antigen. The antigen is injected alone or co-injected with a known adjuvant (such as compound A in the aforementioned examples) or with a potential tolerogen (such as compound G, a pine cone extract). One week following the vaccinatio the mice are adoptively transferred with live syngenic male and female splenocytes that have been differentially labeled with different concentrations of CFSE. At some point following the transfer of live cells (usually 4 hrs) the mice are killed and the relative numbers of CFSE labeled male to female cells determined in the spleen. Higher relative numbers of male cells to female cells, when compared to mice that received antigen alone, is indicative of tolerance indu cti on .

Fu the analysis of Compound G

Using the assay we have identified a compound extracted from pine cones

(referred to as Compound G or PPC) which is a pine cone extract that down- regulates DC function. Utilizing the DC2.4 cell line in the assa we identified several markers of DC function that were down-regulated at the protein level, including MHC-I. We found that PPC down- regulated the levels of β-2 microglobulin (β-2Μ) mRNA suggesting that the effects of PPC on the DCs is mediated at the level of gene expression.

When co-injected into the skin (suhcuianeousiy; s.c.) with ovalbumin protein (OV A), PPC significantly inhibited the functional phenotype of DCs that migrated to the draining lymph node and extinguished their ability to activate OV A-specific T cells.

The effects of PPC on the DC2.4 cells observed in the in vitro assay was mirrored on native DCs in vivo, thereby validating the ability of the assay to detect compounds with the potential to modulate DC function and in particular, inhibit their function.

This appears to be the first demonstration of any compound t inhibit the function of dermal DCs in vivo and may also be the firs demonstration of any compound to inhibit any native (untouched or B DC) DCs in vivo.

PPC modul a tes MHC-I expr es sion a the gene -expr es sion ie vol

β-2ηι is a integral component of the MHC-I complex and β-2ηι expression is necessary for MHC-I expression. We tested whether PPC could affect expression of the p-2m gene, DC2.4 ceils were cultured overnight with OV A (lOO g/mL) in media alone or wit the addition of PPC

( g/mL). The cells were harvested and live cells counted by trypan blue exclusion using a haemocytometer. NA was extracted by NP-40 hypotonic lysis of 3 x. 10 ³ cells and cDNA was prepared. Triplicate cDNA samples were prepared from each culture and each cDNA sample was assayed in duplicate, cDNA was quantified by real-time PCR using primers and probes for β-2Μ, The data shown in Figure. 8 show that PPC down-regulated expression of the β~2πι gene in DC2.4 cells.

P P inkibi t s der mal D functio in vivo

We have recently shown that CT acts directly on dermal DCs when injected s«c. and induces their potent activation and migration to the DLN where they activate T cells. During that study we developed methods to rapidly detect the. effects of a compound on migratory dermal DCs in vivo. As shown in Figure. 9, the migratory DC population in the DLN can be recovered following mechanical disniption of the LN and does not require collagenase treatment or micro-bead enrichment; in fact those commonly used methods, which are necessary to recover the resident DC populations, inhibit the recovery of the migratory DC (in particular the CD 103* subset).

Effects of PPC on DCs following subcutaneous injecti on

We tested the effects of PPC on migratory dermal DCs following s.c. injection as follows: mice i n - 5/ group) were injected in the flank wit OVA (lOOpg) and CT (Ipg) or PPC (l .Smg), control mice received OVA only. 24 h after injection the mice were killed and ipsilateral inguinal and axillar DLNs recovered and single cell suspensions prepared by mechanical disruption. The cells were prepared for FA.CS ^· analysis by first blocking Fc receptors (2.4G2) and then staining with a panel of antibodies to detect the migratory DCs (as shown below in Figure 10).

Injection of PPC had no significant effect on the frequency of the whole migratory DC population; however, the frequency of the CD1 lb ⁺ migratory DC subset was reduced by PPC (Figure 11), The expression of MHC-I and CD86 were not affected on the CD103* subset but were significantly reduced on the CD 103 ^" subset which comprises the CD! lb ⁺ , CD l ib ^" and LC subsets. Of the CD103 ^" subsets, MHC-I expression was reduced on the CDl l b ⁺ and CDllb ^" subsets, but CD86 was only reduced on the CD1 lb ⁺ subset. These data suggest thai PPC most strongly affects the CDllb ⁺ (CD 103 ^" ) subset. These data are a snapshot at 24 h post injection; it is possible that the LC population would also be affected by PPC, however, that population takes longer to extricate from the dermis and arrive at the DLN, so kinetic studies would be required to see if that is the case.

In vi vo PPC exposure severely inhibits the ability of DC to activare _^ C

Our in vitro and in vivo data both suggested that PPC exposure would affect the ability of DCs to activate T cells. We directly tested this by utilizing our s.c. flank injection method as follows: mice were injected s.c. in the flank with OVA protein and PBS or with OVA and the immunomodulatory compounds PPC or CT; 24 h following injection the DLN were recovered and the migratory DCs were sorted into CD103 ⁺ and CD 103 ^" populations and incubated for three days with naive, MACS -purified, CFSE-labeled QT~I CD8 ⁺ T cells which recognize an epitope from OVA in context of MHC-I. Following the incubation the cells are assessed by FACS for viability, number and proliferatio (Figure 12). DCs recovered from mice injected with PBS only (without OVA) failed to induce any measurable proliferation, whereas DCs from mice injected with OVA and PBS or OVA and CT induced strong proliferation, attesting to the sensitivity of the method. In contrast, DCs from mice injected with PPC and OVA had almost completely lost the ability to activate CD8 ⁺ T cells (Figure 13).

The loss of activating potential by the PPC exposed DCs is remarkable. The injection of O A alone without adjuvant was sufficient to induce DCs with very strong activation potential; this suggests that the system is highly sensitive as DCs from mice not injected with OVA had zero activation potential.

It is not clear ho PPC has mediated its effects, PPC may have inhibited the migration of antigen-exposed DCs to the DLN and we can't exclude this without further experiments; however, our phenotypic data shows that DCs in the PPC exposed DLN are inhibited and would certainly have lower activation potential, OVA protem has to be cross-presented to CD8 ⁺ T cells and PPC may also inhibit this pathway, but again, further experiments will be required t see if this is the case. The direct effects of PPC on T cells is unknown, however, our methodology should exclude the potential for PPC to directly contact (he T cells in the assay. The kinetics of the PPC effects also remain unknown and it will be important to know if the inhibition is maintained.

Summary

We have developed a high-throughput assay to determine if a compound can affect DC function. Compounds to be tested are titrated and cultured overnight with human, or mouse DC ceil lines or BMDC in 6-well format plates. The following day, high-throughput multi-parameter flow cytometry i used to assess the effects of the test compound on the DC phenotype and function. Parameters monitored include expression levels of activation and MH.C molecules, che okine and migratory molecules, phagocytosis and cross- presentation as shown in Fig. 7.

We have also performed the assa using a DC cell line and compared ii to primary BMDCs. The DC cell line was more sensitive to the test compounds than the BMDCs and we believe this due to the BMDCs being activated b the process of their generation (which includes culturing with cytokines for more than seven days); whereas, the cell line is grown in media only. Thus, the cell line may be more representative of steady- state (un-activated) in- vivo DCs than BMDCs. Unexpectedly, the assay when used with the cell line was also able to identify compounds with the ability to down-regulate molecules associated with DC activation. This was not anticipated and was not evident when the BMDCs were used - in that case the compounds that inhibited DC function were shown to have little, or no activity - but not an inhibitory (or down-regulatory) effect and, as suggested above, is likely due to the already-activated state of BMDCs.

We have developed simple methods for analyzing the flow -cytometric data and identifying products with the most-potential to modulate DC function in vitro. The next step of the process is to take those lead compounds and test their activity on steady-state DCs in vivo (as in step 4 and 5 of the overview schematic of Fig. 7). Compounds that have in vivo activity confirmed can be further tested in vaccination challenge models, including tumor and infection (step 6 in schematic). Finally, DCs affected by cutaneous injection of the test compound can be recovered from the draining lymph nodes and molecular profiling performed in order to understand the compound's mode of action, An example i microarray analysis such as described in step 7 of Fig. 7,

We identified Compound G/PPC as a potential immunosuppressant in ou dendritic cell assay. Based on previously pubiished reports on pine cone extracts (which are commercially available from man naturopathic suppliers) we were expecting PPC to be an immunostimulant (adjuvant) so it is remarkable and fortuitous that it was tested in our initial studies and that the assay had the unknown potential to identify such a compound as an immunosuppressant with the potential to tolerize DCs . This may be the first demonstration of a method to identify compounds with DC-inhibiting abilities and the first demonstration of such an effect in vivo. It is proposed that the PPC compound may have therapeutic potential.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention t any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure,, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs algorithms, patent and scientific literature referred to herein is incorporated herein by reference in their entirety.