1. Field of the Invention
The present invention relates generally to a model system to identify haematopoietic cells of particular lineages and their stage of differentiation. More particularly, the present invention provides genetically modified cells and non-human animals comprising such cells which carry a genetic marker of terminal differentiation modified to co-produce a reporter molecule capable of eliciting an identifiable signal and their use in identifying molecules capable of modulating the differentiation or transformation status of cells, such as, without limitation, embryonic cells during development, cells with aberrant differentiation such as cancer cells, and cells of the haematopoietic cell lineages such as, for example, B and/or T cells. Identified molecules form the basis for pharmaceutical compositions for therapeutic and prophylactic application.
2. Description of the Prior Art
Bibliographic details of references in the subject specification are also listed at the end of the specification.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Cellular life involves a myriad of alternative and highly regulated biochemical pathways directing changes in cell division, differentiation, morphogenesis and apoptosis. Cells vary in their potential to divide and/or differentiate. For example, the embryo comprises totipotent cells retaining the ability to differentiate into any cell type. Other cell types including stem cells are pluripotent and may ultimately differentiate into a range of but not all cell phenotypes. Some cells become committed to one final form: they are terminally differentiated.
Changes which block normal maturation of cells into terminally differentiated cells or which prevent apoptosis can act as triggers for tumor development characterized by uncontrolled cell division without differentiation or cell death. Thus, agents which promote differentiation and normal apoptosis may switch off tumor development.
Molecules which are expressed during the time of terminal differentiation of particular cell types have been intensely studied. However, in order to understand the sequence of events during this period at a molecular level it is necessary to understand the temporal and spatial expression patterns of molecules which are expressed in this phase of development.
B-lymphocyte-induced maturation factor (Blimp) is a 98 kDa transcription factor which was originally identified as being induced during the differentiation of a B-cell lymphoma cell line (Turner et al., Cell 77:297, 1994). The corresponding factor from human cells is referred to as PRDM-1. It has been proposed that Blimp-1 has a pre-eminent role in regulating B-cell terminal differentiation. Specifically, Blimp-1 is expressed in antibody secreting cells (ASC) from man and mouse but it is not expressed in memory cells (Angelin-Duclos et al., J Immunol 165:5462, 2000). Ectopic expression of Blimp-1 is sufficient to drive terminal differentiation of lymphomas and primary B-cells into ASC cells (Turner et al., (supra), Schliephake et al., Eur J Immunol 26:268, 1996; Messika et al., J Exp Med 188:515, 1998; Knodel et al., Eur J Immunol 31:1972, 2001). Blocking expression of Blimp-1 through antisense or dominant-interfering approaches suppresses cell-cycle exit which is thought to be essential for full ASC differentiation (Soro et al., J Immunol 163:611, 1999; Angelin-Duclos et al., J Immunol 165:5462, 2000; Johnson et al., Eur J Immunol 32:3765, 2002). Also, mice which lack Blimp-1 in B-cells produce very little immunoglobulin and have a markedly reduced ASC compartment. (Shapiro-Shelef et al., Immunity 19:607, 2003).
It was initially reported that Blimp-1 is only produced in cells of the B-cell lineage, however, it is now evident that Blimp-1 is also produced during myeloid differentiation (Keller et al., Genes Dev 5:868, 1991, Chang et al., Nat Immunol 1:169, 2000). Blimp-1 is required for the repression of c-myc which is involved in myeloid differentiation (Chang et al., (supra), 2000; Marcu et al., Annu Rev Biochem 61:809, 1992). Over production of Blimp-1 in U937 cells for example is sufficient to induce macrophage differentiation (Chang et al., (supra), 2000). Thus, repression of c-myc by Blimp-1 in macrophages and B-cells is a feature of terminal differentiation in these two lineages. Blimp-1 is also broadly produced during mouse and Xenopus embryonic development (de Souza et al., Embo J 18:6062, 1999; Rosenbaum et al., Embo J. 9:897, 1990).
B-lymphocytes are among the most intensively studied eukaryotic cell types but while the early steps of B-cell development are relatively well characterized, much less is known about the processes which control the final differentiation of B-lymphocytes into ASC. ASC (plasma cells) are the direct mediators of the humoral immune response. They secrete a large amount of serum immunoglobulin essential for protective immunity. The terminal differentiation of B-lymphocytes into ASC is, therefore, a subject of intense therapeutic interest. For example, terminal differentiation to ASC is a crucial element in effective vaccination strategies. Furthermore, multiple myeloma results from the failure of an ASC to complete the differentiation pathway.
However, ASC represent a very rare population of highly specialised cells located mostly in the bone marrow and spleen. ASC populations in mice and man comprise cells of heterogeneous life span and cell surface phenotype making a definitive prospective isolation of pure ASC impossible (Fong et al., Proc Natl Acad Sci U S A 11:11, 2003; Medina et al., Blood 99:2154, 2002; O'Connor et al., J. Exp Med 195:737, 2002; Manz et al., Curr Opin Immunol 14:517, 2002; Underhill et al., Blood 24:24, 2003).
T-cell terminal differentiation programs are poorly understood (Sprent et al., Immunol Lett 85:145-149, 2003). In response to infection, antigen-specific T cells differentiate into effector cells and undergo massive clonal expansion. Homeostasis of T cell numbers is maintained by the subsequent contraction phase where >90% of effector cells are eliminated with a small fraction becoming memory T cells (Sprent et al., Annu Rev Immunol 20:551-579, 2002). This process has been proposed to be under genetic control as the contraction is independent of the dose or duration of infection (Badovinac et al,, Nat Immunol 5:809-817, 2004; Badovinac et al., Nat Immunol 3:619-626, 2002). The ability to control T cell numbers is essential as enhanced expansion due to the lack of T-regulatory cells (Khattri et al., Nat Immunol 4:337-342, 2003; Hori et al., Science 299:1057-1061, 2003; Fontenot et al,, Nat Immunol 4:330-336, 2003), the loss of the down-regulatory molecule CTLA-4 (Chambers et al., Immunity 7:885-895, 1997) or genetic deficiencies in non-obese diabetic (NOD) mice result in autoimmunity.
The ability to monitor terminal differentiation of ASC, T-cells and other cells of the haematopoietic system in a wide range of contexts and under various stimuli would be extremely valuable in developing strategies and reagents for use in the treatment and/or prophylaxis of a range of conditions associated with aberrant differentiation, such cancer autoimmune disease, or with harnessing normal developmental programs such as in the development of an appropriate immune response.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.
Genes and other genetic material (eg mRNA, constructs etc) are represented in italics and their proteinaceous expression products are represented in non-italicised form. Thus, the transcription factor Blimp is the expression product of Blimp. The term “Blimp” or “Blimp” is used to denote all homologs or variant molecules derived from any animal or mammalian species, including a human homolog. Accordingly, human PRDM-1 and its product, PRDM-1 are encompassed in the terms Blimp or Blimp. Unless otherwise stated, reference to Blimp is a reference to a functional form of the polypeptide and reference to a modified Blimp is a reference to the gene or allele sequences encoding a functional form of Blimp.
The present invention is predicated, in part, on the identification of the role of Blimp in the differentiation of haematopoietic and embryonic cells. By screening for the presence of Blimp, or the level of Blimp, a determination can be made as to the stage of terminal differentiation of a cell. The identification of the role of Blimp further enables substantially homogeneous populations of particular haematopoietic cells to be identified such as, but not limited to, ASC (plasma cells).
More particularly, the present invention provides a genetically modified cell or an in vivo or in vitro system comprising cells which co-express genetic material which encodes Blimp and a reporter molecule. Detection of reporter activity in cells of a haematopoietic lineage, such as but not limited to a lymphocyte lineage, is indicative that cells having reporter activity and producing functional Blimp are committed to terminal differentiation. Thus, the detection of reporter-active B-cells producing functional Blimp is an indication that these cells are committed to differentiate into an antibody secreting cells (ASC). Also, as described herein the detection of reporter activity in T cells expressing a functional Blimp, is indicative that these cells are activated/memory T cells, such as activated CD4 + T-cells or effector CD8 + T-cells. The present invention provides therefore, genetically modified cells or non-human animals comprising such cells which facilitate monitoring the differentiation or transformation status of particular cells under various conditions or in the presence of various stimuli or agents. The present invention further provides screening methods, including high through-put screening methods, for identifying molecules capable of modulating the differentiation or transformation status of cells, such as, without limitation, embryonic cells including stem cells during development, cells with aberrant differentiation such as cancer cells, and cells of the haematopoietic cell lineages such as, for example B and/or T cells.
Specifically, a genetically modified cell, or a non-human organism comprising such cells, is provided by the present invention. In one embodiment, the cells produce Blimp translated from an mRNA modified to encode a reporter molecule. Preferably, the reporter molecule encoding sequence is inserted into an intron of a Blimp allele. When the modified Blimp allele is present in heterozygous form, the other allele will express a functional Blimp. In some embodiments, the modified allele may express a functional Blimp polypeptide or a functional form thereof. In other embodiment the modified allele expressed a non-functional Blimp polypeptide. In one embodiment the modified cells are useful in in vivo or in vitro cellular model systems to identify and isolate, inter alia, ASC. In another embodiment, the modified cells are useful for monitoring the differentiation status of haematopoietic such as T-cells and/or B-cells in a wide range of assays.
In one aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising modified genetic material which when expressed produces a polypeptide co-expressed with a reporter molecule and wherein the polypeptide is associated with terminal differentiation of a haematopoietic cell. Preferably, the genetic material is a Blimp gene or a part, fragment, homolog, derivative or functional form thereof. Furthermore, the identification of the reporter molecule in B-cell lineage cells indicates that such cells are committed to differentiate or have differentiated into ASC. Alternatively, reporter molecule activity in cells of a T cell lineage indicates that these cells are activated. Thus, as described herein, the presence of Blimp in a lymphocyte indicates that the cell is terminally differentiated or is committed to terminal differentiation. Exemplary T-cells include CD4 + T-cells and CD8 + T-cells and exemplary B-cells are ASC. Where a non-functional Blimp polypeptide is produced, detection of reporter-active cells indicates that the cells have been exposed to conditions sufficient to render them terminally differentiated if they had been able to produce a functional Blimp polypeptide.
Genetically modified non-human organisms may be provided in the form of gametes, embryos or ES cells for transplantation. Embryos are preferably maintained in a frozen state and may optionally be sold with instructions for use. Targeting constructs and genetically modified cells are also preferably maintained in a frozen state and may optionally be sold with instructions for use. All such cells are referred to herein as an in vivo or in vitro cellular model system.
The present invention provides a system for monitoring gene expression and differentiation fate in cells in vivo and in vitro at the single cell, tissue and organism level. Thus, reporter activity may be monitored in live cells and gene expression monitored in fixed tissues. Preferably, the reporter expression cassette encodes a fluorescent or other light emitting moiety. The availability of organisms and cells which report the expression of Blimp-1 for example as a marker for terminal differentiation of a particular lineage or cell will be an extremely useful tool in a wide range of applications. In relation to cells of the B-cell lineage, this system finds broad application in the study, isolation and monitoring of ASC. As previously mentioned, ASC have not hitherto been available for study although these cells are crucial for an effective antibody response. Furthermore, aberrant differentiation in ASC causes multiple myeloma in man making them an important cell type to study for this reason.
In a related embodiment, the present invention provides a method for phenotyping and/or monitoring a cell of the haematopoietic system comprising screening a genetically modified cell or non-human animal comprising such cells comprising a modified Blimp gene encoding a Blimp protein which when expressed co-expresses Blimp or a part, fragment, variant, homolog or functional or non-functional form thereof and a reporter molecule, wherein detection of reporter activity is indicative or predictive of a cellular phenotype and/or commitment of a cell to terminally differentiate. Haematopoietic cells include without limitation B-cells, T-cells, dendritic cells, macrophages, natural killer cells, granulocytes, erythrocytes, eosinophils, megakaryocytes, bone marrow, splenic, dermal, or stromal cells or their derivatives. In one particular embodiment, the haematopoietic cells are lymphocytes such as B and/or T cells.
In a further embodiment, cells which exhibit reporter activity or changes in reporter activity are isolated or selected from among cells which do not exhibit reporter activity. Isolation of reporter-active cells may be by any convenient method. For example, flow cytometry, laser scanning cytometry, chromatography and/or other equivalent procedures are conveniently employed. Flow cytometric procedures are particularly preferred. Additionally, further selection markers such as for example drug selection markers, may be used to isolate or select the modified cells of the present invention.
The present invention also provides antagonists and agonists of Blimp-1 expression or Blimp-1 activity. One example of an agonist of Blimp-1 expression is a cytokine such as but not limited to IL-21. Pharmaceutical compositions are further contemplated comprising recombinant, synthetic or isolated forms of the present agonists and antagonists and one or more pharmaceutically acceptable carriers, diluents or excipients. Reference to Blimp-1 expression or production of Blimp-1 protein includes in a single cell or within a population of cells.
| TABLE 1 | |
| Summary of sequence identifiers | |
| SEQUENCE ID | |
| NO: | DESCRIPTION |
| 1 | Nucleotide sequence encoding murine Blimp-1 |
| 2 | Amino acid sequence of murine Blimp-1 |
| 3 | Nucleotide sequence encoding human Blimp-1 |
| (PRDM-1) | |
| 4 | Amino acid sequence of human Blimp-1 (PRDM-1) |
| 5 | Genomic nucleotide sequence of murine Blimp-1 |
| 6 | Genomic nucleotide sequence of human Blimp-1 |
| (PRDM-1) | |
FIG. 1 is a diagrammatic representation of the Blimp-1 locus and a targeting strategy. A) Structural domains of the Blimp-1 protein. The segment of the protein encoded by exons 7-8 are indicated. Acidic, N and C terminal acidic regions; PR, region of homology to the retinoblastoma interacting zinc finger protein RIZ; Pro, proline rich region; Zn, 5 Zinc fingers. B) Genomic locus of Blimp-1, indicating the 8 exons as boxes and introns as black lines. Coding regions are in grey, non-translated regions are white. Restriction enzymes used for Southern hybridisations are marked, along with the 5′ and 3′ probes. Targeted allele derived from the homologous recombination event and subsequent manipulations is indicated C) Southern hybridisation on targeted and control ES cell DNA, using 5′ and 3′ ends of the Blimp-1 locus, to show expected products of the targeting event (4.8 kb 5′ arm and 4.5 kb 3′ arm). Expression of Blimp-1 in blimp gfp/+ LPS stimulated B cells cultured for 0-3 days ex vivo in IL-15 +/− IL-21. Blimp-1 expression was detected using a monoclonal antibody against mouse Blimp-1, a goat polyclonal antibody against α-actin was used as a loading control. +/+ , wild type C57B1/6 mice; −/T blimp gfp/+ mice.
FIG. 2 is a graphical representation showing the results of FACS analysis of Blimp gfp expression in B-cells in vivo. A) Syndecan-1 and Blimp gfp expression in lymph nodes, spleen and bone marrow in Blimp gfp/+ mice (upper panel) and controls (lower panel). B) Expression of Blimp gfp in B220 positive B cells.
FIG. 3 is a graphical and photographic representation showing the results of ELIspot analysis of Blimp gfp sorted cells. Gfp positive cells were sorted from bone marrow (BM) and spleen of an untreated Blimp gfp/+ mouse and analysed in an EliIspot assay. Isotype specific antibodies or anti kappa antibodies were used to coat the elispot plate and to detect secreted immunglobulins. A) Distribution of isotype specific immunglobulins in 200 gfp-positive sorted cells (one representative experiment of three). B) Detection of kappa chain in a single representative well of an ELIspot plate (sample: sorted bone marrow cells). left, input 200 gfp-positive cells; middle, input 100 000 gfp-negative cells; right, input 100 000 unsorted cells.
FIG. 4 is a graphical representation of the results of FACS analysis showing induction of antibody secreting cells with LPS in vivo. Blimp gfp/+ mice were i.v. injected with 2 ug E. coli LPS. Spleens A) and bone marrows B) of these mice were analysed at indicated time points after LPS treatment. LPS induces the formation of ASC, increasing the frequency from about 0.5% to about 5% at day 3 in spleen and from about 0.05% to about 0.25% at day 4 in the bone marrow, respectively, upper panel, FACS scans for syndecan-1 and Blimp gfp , middle panel, syndecan-1 and B220 in gfp-positive gated cells, lower panel, histograms for syndecan-1 and B220 expression in GFP-positive cells at indicated time points.
FIG. 5A is a graphical representation of the kinetics of Blimp gfp expression. Flow cytometry histograms of Blimp gfp expression by stimulated B cells from Blimp gfp/+ mice (red line) and wild type C 57B1/6 mice (blue me) are shown. Histogram gates show a percentage of Blimp gfp positive populations. Highly purified small resting B cells were stimulated recombinant CD40L, IL-4 and IL-5 (top panels) or LPS (20 ug/ml) (bottom panels). Cells were harvested different days of culture time and analysed on flow cytometry. LPS stimulated cells start to express Blimp at 2 days, while in response to CD40L and IL-4/IL-5 Blimp expression become evident 3 days.
FIG. 5B is a graphical representation showing that Blimp gfp positive cells secrete antibodies. Blimp gfp/+ B cells were stimulated with LPS for four days. Cells were harvested and stained with Syndecan-1 (Synd-1) specific antibodies and GFP expressing (left panel, A-C) and non-expressing regions (left panel, D) were sorted directly to the Elispot plates coated with various isotype specific antibodies, using automated cell deposition unit. Sorted cells were processed according to the standart Elispot method. Right panels show number of Ig secreting cells in sorted regions. Most Blimp gfp cells secrete Ig, while all Blimp gfp negative cells do not secrete any of Ig isotypes tested.
FIG. 5C is a graphical representation showing the different expression of Blimp gfp in response to various stimuli. Highly purified small resting B cells were stimulated with i) recombinant CD40L and IL-4; ii) CD40L, IL-4 and IL-5; iii) LPS; iv) LPS and IL-4; v) LPS and anti-IgD monoclonal antibody. After four days of culture cells were harvested, stained with Synd-1 specific antibody and analysed on flow cytometry. Shown here are two parameter dot plots of flow cytometry analysis.
FIG. 6 is a graphical representation showing the results of analyses of mice transplanted with activated B-cells. Purified resting splenic B-cells of Blimp gfp/+ mice were activated for three days in the presence of 20 ug/ml LPS. 3×10 6 cells (containing about 2×10 6 gfp positive cells, i.e. antibody secreting cells, A) were washed three times with LPS and transplanted into WT recipients by i.v. injection. After 7 days the recipient mice were analysed for the presence of donor ASC (B).
FIG. 7A is a tabulated summary of genotyping results of mice born from Blimp gfp/+ ×blimp gfp/+ matings. FIG. 7B is a photographical representation of representative PCR results of genotyping of mice weaned (left) or embryos at day E9.5 (right).
FIG. 8 is a photographic and graphical representation of splenocytes of Blimp gfp/gft and Blimp gfp/+ reconstituted mice were cultured in the presence of 20 ug/ml LPS and analysed for the presence of GFP positive, i.e. antibody secreting cells, at day three (A). GFP positive cells of both cultures were than sorted (B, gate R1) and analysed in an ELIspot assay. While Blimp gfp/+ cells yielded 60-70% antibody secreting cells (B, lower panel left), Blimp gfp/gft gave only 5-7% antibody secreting cells which produced only tiny ELIspot's (B, lower panel, right) compared to spots produced by heterozygous cells. Detection of IgM and kappa chain in single representative wells of an ELISPOT plate (input 200 gfp-positive cells).
FIG. 9 is a graphical representation of the results of FACS analysis of bone marrow derived macrophages (BMM) and blood monocytes. Bone marrow cells were cultured for 7 days in the presence of 10 ng/ml rMCSF, medium was changed and non-adherent cells were removed at day 3 and 5 of culture. Adherent cells (BMM) were analysed for Blimp gfp expression (left panel). Further, MacI/Gr1 double positive blood cells were analysed in FACS (right panel) (black line—wildtype, red line—Blimp gfp/+ ).
FIG. 10 is a graphical representation showing FACS analysis in vitro generated dendritic cells (DC's). Bone marrow cells were cultured for 8 days in the presence of 100 ng/ml Flt3 ligand. Cells were than cultured for another 24 hours (left column) or were stimulated with CpG (1.5 uM), GMCSF (50 ng/ml), gIFN (20 ng/ml) and IL-4 (20 ng/ml) (middle column) or with 1 ug/ml LPS (right column). Blimp gfp expression is shown in a histograms for plasmacytoid DC's and conventional DC's (solid line—wildtype, dotted line—Blimp gfp/+ ).
FIG. 11 is a graphical representation showing FACS analysis of T cells in vivo and in vitro. Thymic (left) and lymph node (middle) T cells, and in vitro activated CD4 + /CD8 + purified lymph node cells (right) of Blimp gfp/+ mice were analysed in FACS. Blimp gfp expression levels of gated T cell populations are shown in histograms (lower panel; black line—wildtype, red line—Blimp gfp/+ ).
FIG. 12 is a graphical representation showing Blimp-1 expression in the NK lineage can be detected in the Blimp gfp/+ reporter mice and induced by maturation stimuli. A) in vivo splenic NK cells are GFP + . B) Sorted NK cells from Blimp gfp/+ spleens were cultured for 4 days in IL-15, followed by 2 days in the indicated cytokine. mfi, mean fluorescence index of Blimp gfp . C) Expression of Blimp-1 in +/+ NK cells cultured for 7 days ex vivo in IL-15 +/− IL-21. Blimp-1 expression was detected using a monoclonal antibody against mouse Blimp-1, a goat polyclonal antibody against α-actin was used as a loading control.
FIG. 13 is a representation showing the cDNA and predicted amino acid sequence of mouse Blimp-1/PRDM-1. The coding sequence is shown in upper case.
FIG. 14 is a representation showing the amino acid sequence of mouse Blimp-1/PRDM-1 derived from the nucleotide sequence (upper case) in FIG. 13.
FIG. 15 is a representation showing the cDNA and predicted amino acid sequence of human Blimp-1/PRDM-1. The coding sequence is shown in upper case.
FIG. 16 is a representation showing the amino acid sequence of human Blimp-1/PRDM-1 derived from the nucleotide sequence (upper case) in FIG. 15.
FIG. 17 is a representation showing the genomic nucleotide sequence of mouse Blimp-1. The genomic locus comprises 8 exons in bold upper case. ATG and stop codons are underlined.
FIG. 18 is a representation showing the genomic nucleotide sequence of human Blimp-1. The genomic locus comprises 8 exons in upper case, bold. ATG and stop codons are underlined.
FIG. 19 is a graphical representation showing that Blimp-1 is expressed in activated/memory T cells, Blimp-1 deficient T cells have an activated/memory phenotype. A) Gated CD4 + or CD8 + splenic T cells of the indicated genotype were examined for GFP fluorescence and activation state. The majority of CD62L low CD4 + T cells and CD44 high CD8 + T cells from Blimp gfp/+ mice is low for GFP, only a small number of CD4 + T cells (CD62L low or high) is GFP high Blimp GFP is strongly expressed in the same population in Blimp gfp/gfp T cells (dot blots); phenotype of Blimp-1 deficient CD 4+ and CD8 + splenic T cells (histograms, solid line blimp-1 +/+ , dotted line blimp-1 gfp/gfp). B) in vitro culture induces Blimp-1 expression. Naïve CD40 + T cells were grown for two rounds in Th1/Th2 polarizing conditions. C) Western blotting for wild type Blimp-1 protein in CD4 + cells grown as above in Th1 or Th2 conditions. Anti-Zap-70 is used as a loading control. B cells stimulated for four days with LPS to induce plasma cell differentiation were used as a positive control. D) Mice reconstituted with the indicated genotypes were analysed post- HSV infection for the appearance of CD8 + T cells specific for the dominant gB 498-505 epitope using a gB specific tetramer. E) in vitro cultured gB-specific CTL show normal cytotoxic function. The HSV infection data is representative of at least three mice of each genotype.
FIG. 20 is a representation of the molecular analysis of Blimp-1 positive CD40 + T cell populations. A) Blimp-1 is expressed in CD25 + suppressor T cells. CD25 + and CD25-CD40 + T cells were sorted (left panel), and naïve CD4 + were differentiated under Th1 and Th2 polarizing conditions (right panel), all populations were subjected to RT-PCR analysis. B) Blimp-1 deficient CD4 + cells secrete high amounts of IFNg and show defective IL-10 secretion. CD 4 + cells were either sorted ex vivo from the spleen and restimulated with plate bound anti CD3 and CD28 for 24 h or differentiated into Th1 or Th2 cells in vitro and subjected to re-stimulation. IL-10 and IFNg in the supernatant was detected in an ELISA.
FIG. 21 is a representation showing that Blimp-1 deficient mice develop a lethal lymphocyte hyperproliferative syndrome. A) Histological examination of Rag1 −/− mice reconstituted with control or Blimp gfp/gfp foetal liver derived stem cells. Blimp gfp/gfp reconstituted mice were sacrificed when moribund. The normal histological appearance of the organs from Blimp gfp/+ mice is contrasted with the lymphocyte infiltration observed in Blimp gfp/gfp mice. B) rapid onset of morbidity in Blimp gfp/gfp but not Blimp gfp/+ reconstituted mice. The number of animals of each genotype examined is indicated.
FIG. 22 is a graphical representation of data showing that Blimp-1 regulates homeostatic proliferation of T cells. A) 3×10 6 Naïve CD4 + or CD8 + splenic T cells from the indicated genotypes were adoptively transferred into non-irradiated Rag2 −/− recipients. Recipients were monitored for weight loss and signs of distress. Graph indicates the percentage changes in weight over the 3-4 week period. 4-6 mice were reconstituted with cells of each genotype. B) Mice that lost >10% body weight were sacrificed and splenic T cell numbers determined. C) splenomegaly of representative Rag2 −/− mice after transfer of Blimp gfp/gfp CD8 + T cells. D) Flow cytometric analysis of Blimp gfp expression in donor T cells 3 weeks after transfer.
FIG. 23 is a representation of data showing that Blimp-1 is induced after secondary stimulation and regulates cytokine responsiveness. A) Naïve CD8 + T cells of the indicated genotypes were cultured in the presence of anti-CD3/CD28 and IL-2 for 5 days before being re-seeded into secondary cultures containing IL-21 for 5 days. B) Identical cell cultures to above were stimulated with anti-CD3/CD28 with or without the indicated cytokine combinations. Total live cell number was determined at the indicated time-points in primary or secondary cultures. CD44 high activated/memory CD8 + cells were subjected only to primary culture.
The present invention is predicated, in part, by the development of a method for identifying and isolating cells of the haematopoietic system or embryonic cells and/or monitoring the differentiation of haematopoietic or embryonic cells, the method comprising detecting or quantifying the presence of a polypeptide (via a reporter) whose presence is associated with terminal differentiation of the cells.
In a particularly preferred embodiment, the polypeptide is Blimp or a part, fragment or functional form thereof which is co-expressed with a reporter molecule.
Accordingly, one aspect of the present invention provides a genetically modified cell or non-human organism comprising such cells comprising genetic material encoding a polypeptide which when expressed produces the polypeptide co-expressed with a reporter molecule and which polypeptide is associated with a cellular phenotype including a commitment in the cell to terminally differentiate.
In a further aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp polypeptide which when expressed produces Blimp or a part, fragment or functional form thereof co-expressed with a reporter molecule and wherein the presence of Blimp is associated with a cellular phenotype including a commitment in the cell to terminally differentiate.
In a further preferred aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence, wherein the presence of Blimp is associated with a cellular phenotype including a commitment in the cell to terminally differentiate.
Preferably, the reporter molecule encoding sequence is inserted with an intron of the Blimp allele. In this way, the modified Blimp allele co-produces the reporter from a bicistronic RNA under the control of endogenous Blimp regulatory elements.
The terms “co-expression” and “co-production” are used herein in a broad sense to refer to the transcription of two or more nucleic acid regions (expressed as one or more RNAs) at the same time or at substantially the same time and their subsequent translation (produced as one or more polypeptides) at the same or substantially the same time. Preferably, one transcript is expressed which encodes both Blimp or a part, fragment or functional form thereof and a reporter molecule. In each case, the expression of the reporter is operatively linked to the expression of the molecule to be reported.
Reference to “cellular phenotype” herein encompasses the molecular or functional characteristics of a cell. For example, ASC cells express Blimp-1 (a molecular marker) and are functionally distinguished from other B-cells by exhibiting, inter alia, a high rate of Ig secretion, the absence of MHC class II molecules and low levels of surface Ig. As used herein, the term is a reference to the full range of molecular or functional characteristics, or any particular molecules or functional characteristic in addition to the molecular characteristic of modulated levels of Blimp-1 expression.
The genetically modified cell or non-human organism comprising such cells may comprise cells or genetic material from any organism such as, but not limited to, humans, non- human primates, livestock, companion or laboratory test organism, reptilian or amphibian species. Preferably the genetically modified organism is a mouse or other laboratory test animal such as a rat, guinea pig, pig, rabbit or sheep.
As used herein the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” includes a single cell, as well as two or more cells; reference to “a gene” includes a gene, as well as two or more gene; and so forth.
The modified gene of the present invention is a marker for terminal differentiation in cells of the haematopoietic system, such as B-cell lineage cells.
Reference to a “genetically modified cell” is a reference to any cell which has been engineered to comprise a sequence of nucleotides from a coding or non-coding region of the genome which is altered relative to its pre-modified form, and its progeny. In particular, the cell is genetically modified to co-express a genetic marker of terminal differentiation and a reporter molecule encoding sequence. Preferably, the cell is genetically modified to co-express Blimp or a part, fragment or functional part thereof and a reporter molecule. The reporter molecule may be any molecule capable of directly or indirectly providing an identifiable signal. A fluorescent or other light emitting reporter molecule is particularly preferred.
Conveniently, targeting constructs are initially used to generate the modified genetic sequences in the cell or organism. Targeting constructs generally but not exclusively modify a target sequence by homologous recombination. Alternatively, a modified genetic sequence may be introduced using artificial chromosomes. Targeting or other constructs are produced and introduced into target cells using methods well known in the art which are described in molecular biology laboratory manuals such as, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Edition, CSHLP, CSH, NY, 2001; Ausubel (Ed) Current Protocols in Molecular Biology, 5th Edition, John Wiley & Sons, Inc, NY, 2002. Targeting constructs may be introduced into cells by any method such as electroporation, viral mediated transfer or microinjection. Selection markers are generally employed to initially identify cells which have successfully incorporated the targeting construct. As the skilled artisan will appreciate, the subject modified organisms may be genetically modified to express the Blimp allele and reporter molecule in only certain cells.
In one particular embodiment the present invention provides a nucleic acid construct suitable for use as a targeting construct said construct comprising all or a portion of an allele of Blimp-1 and a reporter construct. The construct comprise genetic material which encodes a functionally active Blimp-1 polypeptide or a functionally inactive Blimp-1 polypeptide. In a particular embodiment, the construct encodes a partial Blimp-1 polypeptide which lacks a zinc finger domain comprising a DNA binding motif. In a particularly preferred embodiment, the construct is flanked by sites to facilitate recombinase mediated deletion and homologous recombination of the nucleic acid construct into a target genetic sequence. Alternatively, the construct may be introduced into a host cell where it replicates episomally.
Genetically modified organisms are generated using techniques well known in the art such as described in Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbour Laboratory Press, CSH NY, 1986; Mansour et al., Nature 336:348-352, 1988; Pickert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press, San Diago, Calif., 1994. Stem cells including embryonic stem cells (ES cells) are introduced into the embryo of a recipient organism at the blastocyst stage of development. There they are capable of integration into the inner cell mass where they develop and contribute to the germ line of the recipient organism. ES cells are conveniently obtained from pre-implantation embryos maintained in vitro (Robertson et al., Nature 322:445-448, 1986). Once correct targeting has been verified, modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism. Alternatively, modified cells are allowed to aggregate with dissociated embronic cells to form aggregation chimera. The chimeric organism is then implanted into a suitable female foster organism and the embryo allowed to develop to term. Chimeric progeny are bred to obtain offspring in which the genome of each cell contains the nucleotide sequences conferred by the targeting construct. Genetically modified organism may comprise a heterozygous modification or alternatively both alleles may be affected.
In accordance with the present invention it is surprisingly determined that Blimp-1 is essential for the production of antibody by ASC but not the commitment to differentiate down the ASC pathway. Accordingly, the identification of Blimp (eg via a reporter molecule co-expressed therewith) in B-cell lineage cells indicates that the cells are committed to differentiate or have differentiated into ASC.
Furthermore, as disclosed herein, Blimp is essential for lymphocyte homeostasis including T-cell homeostasis and the ability of T-cells to become terminally differentiated. The absence of Blimp in adult mammals leads to aggressive multi-organ lymphproliferative disease.
Accordingly, another aspect of the present invention provides a genetically modified cell or non-human organism comprising such cells comprising genetic material encoding a polypeptide which when expressed produces the polypeptide co-expressed with a reporter molecule wherein detection of said reporter molecule is indicative of a cellular phenotype and/or commitment of a cell to terminally differentiate.
In a further aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp polypeptide which when expressed produces Blimp or a part, fragment or functional form thereof co-expressed with a reporter molecule and wherein detection of said reporter molecule is indicative of a cellular phenotype and/or commitment of a cell to terminally differentiate.
In a further preferred aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence, wherein detection of said reporter molecule is indicative of a cellular phenotype and/or commitment of a cell to terminally differentiate
Preferably, the reporter molecule encoding sequence is inserted with an intron of the Blimp allele. In this way, the modified Blimp allele co-produces the reporter from a bicistronic RNA under the control of endogenous Blimp regulatory elements.
Accordingly, another aspect of the present invention provides a genetically modified cell or non-human organism comprising such cells comprising genetic material encoding a polypeptide which when expressed produces the polypeptide co-expressed with a reporter molecule and wherein detection of said reporter molecule in cells of the haematopoietic system is indicative of a cellular phenotype and/or commitment of a cell to terminally differentiate.
In a further aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp polypeptide which when expressed produces Blimp or a part, fragment or functional form thereof co-expressed with a reporter molecule and wherein detection of said reporter molecule in B-cells is indicative that cells having reporter molecule activity are committed to differentiation into ASC.
In a further preferred aspect, the present invention provides a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene encoding a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence, wherein detection of said reporter molecule in T-cells is indicative that cells having reporter molecule activity are activated T-cells.
Preferably, the reporter molecule encoding sequence is inserted with an intron of the Blimp allele. In this way, the modified Blimp allele co-produces the reporter from a bicistronic RNA under the control of endogenous Blimp regulatory elements.
Reference herein to a Blimp-1 gene or nucleic acid expression product thereof (RNA) includes homologs, parts, fragments, functional forms thereof including functional variants or derivatives which hybridize thereto under low stringency conditions or comprise significant sequence similarity to all or a functional part such as at least about 60% sequence similarity, after optimal alignment. Reference to a Blimp-1 polypeptide or protein is used in a broad sense to include all homologs, parts, fragments or functional forms thereof including functional variants or derivatives bearing at least about 60% amino acid sequence similarity after optimal alignment.
Functional parts of the instant molecules include portions of the full length molecule which are important for the particular functions thereof such as substrate binding, tertiary conformation or transcriptional activity. Transcription initiation sites are readily mapped and sites conferring promoter activity readily identified (see for example Tunyaplin et al., Nucleic Acid Research 28(24):4846-4855, 2000). Functional parts are important for regulating the expression and activity of the molecule. Functional variants or derivatives retain at least one of the functional activities important for regulating expression and activity of a reference molecule. With reference to Blimp-1, its expression is associated with terminal differentiation, induction of Ig secretion by ASC cells and activation of T-cells.
The modified Blimp gene may encode a functionally active Blimp polypeptide, a functionally inactive Blimp polypeptide and/or partial Blimp polypeptide such as a polypeptide or peptide, for example, lacking a zinc finger domain comprising a DNA binding motif. The terms “polypeptide” and “protein” are used interchangeably herein.
A “part” in peptide form may be as small as an epitope comprising less than 5 amino acids or as large as several hundred kilodaltons. The length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues. A “part” of a nucleic acid molecule is defined a having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.
The present invention also contemplates modified Blimp alleles encoding variant Blimp polypeptides. “Variant” polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein (i.e, they are transcriptional repressors of for example c-myc and/or CIITA). Alternatively, the variant Blimp polypeptides are non-functional. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native Blimp polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a Blimp polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
A Blimp polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a Blimp polypeptide can be prepared by mutations in the encoding nucleic acid sequence. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel ( Proc. Natl. Acad. Sci. USA 82:488-492, 1985), Kunkel et al., ( Methods in Enzymol. 154:367-382, 1987), U.S. Pat. No. 4,873,192, Watson et al. (“Molecular Biology of the Gene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do or do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., ( Natl. Biomed. Res. Found 5:345-358, 1978). For example deletion of all or part of the zinc finger domains containing the DNA binding motif will produce a Blimp variant which is functionally inactive. In some embodiments, animal models are heterozygous in some or all tissues for a genetically modified non-functional Blimp allele, while the other allele comprises a functional Blimp allele capable of expressing a functional Blimp polypeptide. In other embodiments, homozygous animals are produced which do not express Blimp in particular cells or tissues. Alternatively functional Blimp may be produced by one or two modified Blimp alleles in a cell, tissue or non-human organism. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of Blimp polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify Blimp polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA 89:7811-7815, 1992; Delgrave et al. Protein Engineering 6:327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.
Variant Blimp polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to the parent Blimp amino acid sequence. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.
Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.
Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
This description also characterises certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, “small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., 1978 (supra); and by Gonnet et al., Science 256(5062):1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a “small” amino acid.
The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.
Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table A.
| TABLE A | |
| Amino acid sub-classification | |
| Sub-classes | Amino acids |
| Acidic | Aspartic acid, Glutamic acid |
| Basic | Noncyclic: Arginine, Lysine; Cyclic: Histidine |
| Charged | Aspartic acid, Glutamic acid, Arginine, Lysine, |
| Histidine | |
| Small | Glycine, Serine, Alanine, Threonine, Proline |
| Polar/neutral | Asparagine, Histidine, Glutamine, Cysteine, Serine, |
| Threonine | |
| Polar/large | Asparagine, Glutamine |
| Hydrophobic | Tyrosine, Valine, Isoleucine, Leucine, Methionine, |
| Phenylalanine, Tryptophan | |
| Aromatic | Tryptophan, Tyrosine, Phenylalanine |
| Residues that | Glycine and Proline |
| influence | |
| chain orientation | |
Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional Blimp polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table B below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
| TABLE B | ||
| Exemplary and Preferred Amino Acid Substitutions | ||
| Orginal | PREFERRED | |
| Residue | EXEMPLARY SUBSTITUTIONS | SUBSTITUTIONS |
| Ala | Val, Leu, Ile | Val |
| Arg | Lys, Gln, Asn | Lys |
| Asn | Gln, His, Lys, Arg | Gln |
| Asp | Glu | Glu |
| Cys | Ser | Ser |
| Gln | Asn, His, Lys, | Asn |
| Glu | Asp, Lys | Asp |
| Gly | Pro | Pro |
| His | Asn, Gln, Lys, Arg | Arg |
| Ile | Leu, Val, Met, Ala, Phe, Norleu | Leu |
| Leu | Norleu, Ile, Val, Met, Ala, Phe | Ile |
| Lys | Arg, Gln, Asn | Arg |
| Met | Leu, Ile, Phe | Leu |
| Phe | Leu, Val, Ile, Ala | Leu |
| Pro | Gly | Gly |
| Ser | Thr | Thr |
| Thr | Ser | Ser |
| Trp | Tyr | Tyr |
| Tyr | Trp, Phe, Thr, Ser | Phe |
| Val | Ile, Leu, Met, Phe, Ala, Norleu | Leu |
Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid., aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm. C. Brown Publishers (1993).
Thus, a predicted non-essential amino acid residue in a Blimp polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a Blimp polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
Accordingly, the present invention also contemplates variants of the naturally-occurring Blimp polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% similarity to a parent Blimp polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2 and 4. Desirably, variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a reference Blimp polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2 and 4. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which retain the properties of the parent Blimp polypeptide are contemplated. Blimp polypeptides also include polypeptides that are encoded by polynucleotides that hybridise under stringency conditions as defined herein, especially high stringency conditions, to Blimp polynucleotide sequences, or the non-coding strand thereof.
In some embodiments, variant polypeptides differ from an Blimp sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 2 and 4 by at least 1% but less than 20%, 15%, 10% or 5% of the residues, (If this comparison requires alignment the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of an Blimp polypeptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.
In other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a Blimp polypeptide as, for example, set forth in any one of SEQ ID NO: 2 and 4.
The present invention encompasses Blimp from any mammal or animal (including avian species) subject such as from humans, non-human primates, livestock, laboratory, companion or wild animals. Reference to “Blimp” includes Blimp or Blimp from any of the above species as well as structural or evolutionary equivalents or homologs thereof. for example, the present invention encompasses Blimp or a Blimp having an amino acid sequence which has substantially at least about 60% similarity to SEQ ID NO: 2 or 4 or at least about 60% identity to SEQ ID NO: 1, 3, 5 or 6. Reference to at least about 60% includes 60, 61, 62, 63, 64% and all following consecutive numbers in the series to 100%.
Function derivatives of molecules in nucleic acid form include nucleic acid molecules comprising a nucleotide sequence capable of hybridising to the molecule or its complementary form under low stringency conditions.
The terms “similarity” or identity as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7 . 0 , Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al., Nucl. Acids Res. 25: 3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology John Wiley & Sons Inc, 1994-1998, Chapter 15).
The terms “sequence similarity” and “sequence identity” as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
Furthermore, a Blimp homolog or derivative may be defined as being capable of hybridising to SEQ ID NO: 1, 3, 5 or 6 or to a complementary form thereof under low stringency conditions.
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T m =69.3+0.41 (G+C)% (Marmur et al., J. Mol. Biol. 5: 109, 1962). However, the T m of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (Bonner et al., Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
Preferably the modified Blimp gene is modified using a nucleic acid construct comprising all or a portion of an allele of Blimp into which a nucleotide sequence encoding a reporter molecule is inserted.
The reporter molecule is conveniently encoded by a reporter expression cassette or reporter construct. The reporter construct can be brought under the control of the Blimp-1 regulatory elements and faithfully report the Blimp-1 expression pattern in cells, tissues or organisms.
By “reporter” is meant any molecule, protein or polypeptide which is typically encoded by a reporter gene and measured in a reporter assay. Reporters provide a detectable signal which permit an understanding of the activity of genetic sequences. They may report an activity directly or may indirectly monitor activity by monitoring the activity of down stream targets. A reporter protein should be distinguishable from other proteins and ideally, readily quantified. The reactivity between an epitope and an antibody determined thereby may readily be employed optionally together with second or further antibodies. Common reporter proteins include luciferase, chloramphenicol transferase (CAT), Beta-galactosidase (B-gal), or fluorescent proteins such as green fluorescent proteins (GFP). Reference herein to GFP is meant to encompass any fluorescent or light-emitting protein including those derived from jellyfish or other organisms and all homologues, derivatives, analogues including colour variants such as DSRed, HcRed, Clontech; or hrGFP, Stratagene). Preferably said reporter expression cassette encodes a fluorescent or other light emitting GFP. GFP reporters are readily detectable in live cells and are particularly useful and preferred in cell sorting applications.
Examples of fluorescent or light emitting markers may be selected from among those included, but are not limited to those, in the following Table 2.
| TABLE 2 | ||
| Probe | Ex 1 (nm) | Em 2 (nm) |
| Reactive and conjugated probes | ||
| Hydroxycoumarin | 325 | 386 |
| Aminocoumarin | 350 | 455 |
| Methoxycoumarin | 360 | 410 |
| Cascade Blue | 375; 400 | 423 |
| Lucifer Yellow | 425 | 528 |
| NBD | 466 | 539 |
| R-Phycoerythrin (PE) | 480; 565 | 578 |
| PE-Cy5 conjugates | 480; 565; 650 | 670 |
| PE-Cy7 conjugates | 480; 565; 743 | 767 |
| APC-Cy7 conjugates | 650; 755 | 767 |
| Red 613 | 480; 565 | 613 |
| Fluorescein | 495 | 519 |
| FluorX | 494 | 520 |
| BODIPY-FL | 503 | 512 |
| TRITC | 547 | 574 |
| X-Rhodamine | 570 | 576 |
| Lissamine Rhodamine B | 570 | 590 |
| PerCP | 490 | 675 |
| Texas Red | 589 | 615 |
| Allophycocyanin (APC) | 650 | 660 |
| TruRed | 490, 675 | 695 |
| Alexa Fluor 350 | 346 | 445 |
| Alexa Fluor 430 | 430 | 545 |
| Alexa Fluor 488 | 494 | 517 |
| Alexa Fluor 532 | 530 | 555 |
| Alexa Fluor 546 | 556 | 573 |
| Alexa Fluor 555 | 556 | 573 |
| Alexa Fluor 568 | 578 | 603 |
| Alexa Fluor 594 | 590 | 617 |
| Alexa Fluor 633 | 621 | 639 |
| Alexa Fluor 647 | 650 | 688 |
| Alexa Fluor 660 | 663 | 690 |
| Alexa Fluor 680 | 679 | 702 |
| Alexa Fluor 700 | 696 | 719 |
| Alexa Fluor 750 | 752 | 779 |
| Cy2 | 489 | 506 |
| Cy3 | (512); 550 | 570; (615) |
| Cy3,5 | 581 | 596; (640) |
| Cy5 | (625); 650 | 670 |
| Cy5,5 | 675 | 694 |
| Cy 7 | 743 | 767 |
| Nucleic acid probes | ||
| Hoeschst 33342 | 343 | 483 |
| DAPI | 345 | 455 |
| Hoechst 33258 | 345 | 478 |
| SYTOX Blue | 431 | 480 |
| Chromomycin A3 | 445 | 575 |
| Mithramycin | 445 | 575 |
| YOYO-1 | 491 | 509 |
| SYTOX Green | 504 | 523 |
| SYTOX Orange | 547 | 570 |
| Ethidium Bormide | 493 | 620 |
| 7-AAD | 546 | 647 |
| Acridine Orange | 503 | 530/640 |
| TOTO-1, TO-PRO-1 | 509 | 533 |
| Thiazole Orange | 510 | 530 |
| Propidium Iodide (PI) | 536 | 617 |
| TOTO-3, TO-PRO-3 | 642 | 661 |
| LDS 751 | 543; 590 | 712; 607 |
| Cell function probes | ||
| Indo-1 | 361/330 | 490/405 |
| Fluo-3 | 506 | 526 |
| DCFH | 505 | 535 |
| DHR | 505 | 534 |
| SNARF | 548/579 | 587/635 |
| Fluorescent Proteins | ||
| Y66F | 360 | 508 |
| Y66H | 360 | 442 |
| EBFP | 380 | 440 |
| Wild-type | 396, 475 | 50, 503 |
| GFPuv | 385 | 508 |
| ECFP | 434 | 477 |
| Y66W | 436 | 485 |
| S65A | 471 | 504 |
| S65C | 479 | 507 |
| S65L | 484 | 510 |
| S65T | 488 | 511 |
| EGFP | 489 | 508 |
| EYFP | 514 | 527 |
| DsRed | 558 | 583 |
| Other probes | ||
| Monochlorobimane | 380 | 461 |
| Calcein | 496 | 517 |
| 1 Ex: Peak excitation wavelength (nm) | ||
| 2 2Em: Peak emission wavelength (nm) | ||
Any suitable method of analyzing fluorescence emission is encompassed by the present invention. In this regard, the invention contemplates techniques including but not restricted to 2-photon and 3-photon time resolved fluorescence spectroscopy as, for example, disclosed by Lakowicz et al., Biophys. J. 72: 567, 1997, fluorescence lifetime imaging as, for example, disclosed by Eriksson et al., Biophys. J. 2: 64, 1993, incorporated herein by reference) and fluorescence resonance energy transfer as, for example, disclosed by Youvan et al., Biotechnology et Elia 3: 1-18, 1997).
Exemplary fluorophores which may be used in accordance with the present invention include those discussed by Dower et al. (International Patent Publication No. WO 93/06121). Preferably, fluorescent dyes are employed. Any suitable fluorescent dye may be used for incorporation into the instant reporter molecule. For example, reference may be made to U.S. Pat. No. 5,573,909 (Singer et al.) and U.S. Pat. No. 5,326,692 (Brinkley et al.) which describe a plethora of fluorescent dyes. Reference may also be made to fluorescent dyes described in U.S. Pat. Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,986, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,515,864, 5,648,270 and 5,723,218.
A modern flow cytometer is able to perform these tasks up to 100,000 cells/particles s −1 . Through the use of an optical array of filters and dichroic mirrors, different wavelengths of fluorescent light can be separated and detected simultaneously. In addition, a number of lasers with different excitation wavelengths may be used. Hence, a variety of fluorophores can be used to target and examine, for example, intra- and extra-cellular properties of individual cells. The scattered light measurements can also classify an individual cells's size, shape, granularity and/or complexity and, hence, belonging to a particular population of interest (Shapiro, Practical flow cytometry, 3 rd Ed., Brisbane, Wiley-Liss, 1995).
Suitable flow cytometers which may be used in the methods of the present invention include those which measure five to nine optical parameters (see Table 3) using a single excitation laser, commonly an argon ion air-cooled laser operating at 15 mW on its 488 nm spectral line. More advanced flow cytometers are capable of using multiple excitation lasers such as a HeNe laser (633 nm) or a HeCd laser (325 nm) in addition to the argon ion laser (488 or 514 nm). Optical parameters, corresponding to different optically detectable/quantifiable attributes, for a carrier, may be measured by a flow cytometer to provide a matrix of qualitative and/or quantitative information, providing a code (or addressability in a multi-dimensional space) for the carrier.
For example, Biggs et al. ( Cytometry 36: 36-45, 1999) have constructed an 11-parameter flow cytometer using three excitation lasers and have demonstrated the use of nine distinguishable fluorophores in addition to forward and side scatter measurements for purposes of immunophenotyping (i.e. classifying) cells. The maximum number of parameters commercially available currently is 17: forward scatter, side scatter and three excitation lasers each with five fluorescence detectors. Whether all of the parameters can be adequately used depends heavily on the extinction coefficients, quantum yields and amount of spectral overlap between all fluorophores (Malemed et al., “ Flow cytometry and sorting ”, 2 nd Ed., New York, Wiley-Liss, 1990). However, it will be understood that the present invention is not restricted to any particular flow cytometer or any particular set of parameters. In this regard, the invention also contemplates use in place of a conventional flow cytometer, a microfabricated flow cytometer as, for example., disclosed by Fu et al., Nature Biotechnology 17: 1109-1111, 1999.
| TABLE 3 | |||
| Exemplary optical parameters which may be measured | |||
| by a flow cytometer. | |||
| Detection angle form | Wavelength | ||
| Parameter | Acronym | incident laser beam | (nm) |
| Forward scattered light | FS | 2-5° | 488* |
| Side scattered light | SS | 90° | 488* |
| “Green” fluorescence | FL1 | 90° | 510-540 † |
| “Yellow” fluorescence | FL2 | 90° | 560-580 † |
| “Red” fluorescence | FL3 | 90° | >650 # |
| *using a 488 nm excitation laser | |||
| † width of bandpass filter | |||
| # longpass filter | |||
A flow cytometer with this capacity to sort is known as a “fluorescence-activated cell sorter” (FACS). Accordingly, the step of sorting in the present method of obtaining a population of detectably unique carriers may be effected by flow cytometric techniques such as by fluorescence activated cell sorting (FACS) although with respect to the present invention, FACS is more accurately “fluorescence activated carrier or solid support sorting” (see, for example, “ Methods in Cell Biology ” Vol. 33, Darzynkiewica, Z. and Crissman, H. A., eds., Academic Press).
In a further embodiment the present invention provides a method for phenotyping and/or monitoring a cell of the haematopoietic system comprising screening a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene wherein expression or activity of said gene is indicative of a cellular phenotype and/or a commitment of said cell to terminally differentiate. Haematopoietic cells include but are not limited to B-cells, T-cells, dendritic cells, macrophages and natural killer cells, granulocytes, eosinophils, erythrocytes, megakaryocytes, bone marrow, stromal, splenic precursor cells and their derivatives.
Preferably the modified Blimp gene encodes a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence which when expressed produces Blimp or a part, fragment or functional form thereof co-expressed with a reporter molecule and wherein detection of the reporter molecule is indicative of cellular phenotype and/or commitment of a cell to terminally differentiate.
In a further embodiment, cells which exhibit reporter activity or changes in reporter activity are isolated or selected from among cells which do not exhibit reporter activity. Isolation of reporter-active cells may be by flow cytometry, laser scanning cytometry, chromatography and/or other equivalent procedures. Additionally, further selection markers may be used to isolate or select the modified cells of the present invention. Flow cytometric isolation is particularly preferred.
Preferably the cells are ASC identified or isolated in a population of cells of a B-cell lineage.
Accordingly, the present invention provides a method for isolating a substantially purified population of ASC from a population of substantially B-cells said method comprising contacting a genetically modified cell or non-human organism comprising such cells comprising a modified Blimp gene with an agent or composition capable of inducing differentiation to ASC wherein expression or activity of said gene is reported by a reporter construct and wherein detection of said reporter activity is indicative that cells with reporter molecule activity are ASC, where necessary isolating B-cells from said organism and isolating ASC based on the activity of the reporter molecule.
Preferably the modified cell comprises a modified Blimp gene encoding a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence which when expressed produces Blimp or a part, fragment or functional form thereof co-expressed with a reporter molecule and wherein reporter activity is indicative that cells with reporter molecule activity are ASC.
Preferably, screening of cells is achieved by flow cytometric analysis of a fluroescent reporter molecule.
B-cells are conveniently isolated from an organism or sample for example by density gradient centrifugation, flow cytometry or using magnetic beads. Any agent or composition which selectively, clonally or polyclonally of otherwise effectively activates B-cells and induces their differentiation to ASC is encompassed. An example of a polyclonal activator is LPS.
In one embodiment the reporter is a GFP and said ASC are isolated by flow cytometry.
Substantially purified means that the ASC comprise at least about 60 to 95%, preferably at least about 97%, more preferably at least about 99% of the cells, such as at least about 60, 61, 62, 63, 64 and following subsequent numbers in the series to 100%. Alternatively, enrichment of approximately 100,000 fold over unsorted cells is contemplated.
The present invention also provides a method for testing the antigenicity of a vaccine or the ability of agents to enhance or suppress antibody production by ASC wherein reduced reporter activity is indicative of an agent which down regulates or inhibits an antibody response and reporter activity or enhanced reporter activity relative to controls is indicative of agents which are positive regulators of the antibody response. In accordance with this aspect, the method comprises:
In another embodiment, the present invention provides a method for testing the antigenicity or immunogenicity of a vaccine comprising a genetic or proteinaceous composition, the method comprising;
In some embodiments, the Blimp gene encodes a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule coding sequence. In other embodiments, the reporter molecule coding sequence is inserted within an intron of a Blimp allele. In further embodiments, the modified Blimp allele is present in homozygous or heterozygous form. Depending upon the purpose of the assay in some embodiments, the modified Blimp allele encodes a functional Blimp transcription factor or a functional part, form, homolog or variant thereof. In other embodiments, the modified Blimp allele encodes a non-functional Blimp transcription factor or a non-functional part, form, homolog or variant thereof. In an illustrative embodiment, the cells or genetic material are derived from man, a non-human primate, a livestock, companion or laboratory test organisms, reptilian or amphibian species. Examples of laboratory test animal include a rodent (including mice), guinea pig, pig, duck, rabbit or sheep.
In some illustrative embodiments of the methods disclosed herein the cell is a haematopoietic or embryonic cell. As disclosed herein Blimp is essential for both B-cell and T-cell terminal differentiation and accordingly a preferred cell lineage is is a lymphocytic cell. In particular embodiments the lymphocytic cell types is selected from a B-cell and a T-cell. Where a B-cell, the terminally differentiated form is an ASC and these cell can furthermore be substantially purified using the methods disclosed herein. When the cell is a B-cell, the terminally differentiated T-cells include without limitation CD40 + T-cells and CD8 + T-cells. Conveniently, the detection of the reporter molecule is indicative or predictive of a cellular phenotype and/or commitment of a cell to terminally differentiate under particular conditions or in the presence of test agents. Still more conveniently, the reporter molecule is a fluorescent or light emitting reporter molecule.
The present invention also directed to antagonists and agonists of terminal differentiation of cells such as, but not limited to ASC including antagonists and agonists of Blimp-1 expression or Blimp-1 activity, identified by the herein described method, for use in modulating cellular differentiation, The molecules to which the instant modulators, agonists or antagonists are directed are collectively referred to herein as “targets” or “target molecules”.
In another aspect therefore, the present invention provides methods for in vitro or in vivo screening for agonists or antagonists of terminal differentiation in haematopoietic cells comprising exposing one or more agent/s to a genetically modified cell or non-human animal comprising such cells wherein the cell or organism comprises a modified Blimp-1 gene which encodes a Blimp polypeptide which when expressed produces Blimp or a part or fragment or functional form thereof co-expressed with a reporter molecule; and testing the cell or organism for the presence or a change in the level of the reporter molecule the presence of which is indicative of the ability of the one or more agent/s to agonise or antagonise terminal differentiation. Agonists of Blimp directly or indirectly induce terminal differentiation of haematopoietic cells and are useful, for example, in the treatment or prevention of cancer and/or autoimmune disease and in promoting appropriate immune responses to pathological infections. Molecules which inhibit generation of terminally differentiated cells are useful in autoimmune patents such as lupus patients or in treating immune dysfunction such as cases of allegy.
Preferably the modified cell is a haematopoietic cell which comprises a modified Blimp gene encoding a Blimp mRNA transcript comprising a Blimp coding sequence or a part, fragment or functional form thereof and a reporter molecule encoding sequence which when expressed produces Blimp or a part, fragment, homolog, variant, derivative or functional or non-functional form thereof co-expressed with a reporter molecule and wherein reporter activity is indicative that cells with reporter molecule activity are terminally differentiated or committed to terminal differentiation. More preferably, the haematopoietic cell is a lymphocyte lineage cell. In some embodiments the terminally differentiated cells are ASC: in other embodiments, the terminally differentiated cells are CD4 T-cell and/or CD8 T-cells. The modified Blimp allele is present in the cell, tissue or non-human organism in homozygous or heterozygous form. Furthermore, depending upon the particular application, the Blimp allele expresses a transcriptionally (functionally) active Blimp polypeptide. Thus in some assays it will be useful to have a functional Blimp polypeptide to modulate or induce terminal differentiation in a cell. In other embodiments, the modified Blimp allele does not express a functional Blimp and it will be sufficient to determine, via detection of the reporter activity whether a Blimp allele would have been expressed or whether the level of Blimp expression would have been modulated in a cell capable of producing a function Blimp polypeptide.
Cellular (in vitro) assays are particularly convenient and, when coupled with a reporter molecule whose activity can readily be detected in cells, the assays are ideally suited to high throughput screening. A large number of different formats are available as known to the skilled artisan. One useful example is described in Ulleras et al., Toxicology 206(2):245-256, 2005.
“Modulation” of a molecule or differentiation status includes completely or partially inhibiting or reducing or down regulating all or part of its functional activity or differentiation and enhancing or up regulating all or part its functional activity or differentiation. Where the molecule is a genetic sequence its functional activity may be modulated by, for example, modulating its binding capabilities or transcriptional or translational activity, or its half-life. Where the molecule is an encoded polypeptide, its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell or membrane or its enzymatic capability. Modulators are agonists or antagonists which achieve modulation. Enhanced differentiation can also be indicative of reduced cell division.
An example of an antagonist or agonist is a protein, polypeptide or peptide. These terms may be used interchangeably. These terms refer to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, polypeptides, peptides, oligopeptides and proteins are included within the one definition of a polypeptide. These terms also do not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as those given in Table 4 or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell. Polypep