WO2004047860A2 - Cellules non presentatrices d'antigene pour supprimer des reactions immunitaires pathologiques - Google Patents

Cellules non presentatrices d'antigene pour supprimer des reactions immunitaires pathologiques Download PDF

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WO2004047860A2
WO2004047860A2 PCT/EP2003/013402 EP0313402W WO2004047860A2 WO 2004047860 A2 WO2004047860 A2 WO 2004047860A2 EP 0313402 W EP0313402 W EP 0313402W WO 2004047860 A2 WO2004047860 A2 WO 2004047860A2
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cells
cell
ciita
mhc
antigens
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Ahmed Sheriff
Birgit Vogt
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Genethor Gmbh
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the use of non-antigen-presenting cells for the in vivo selection of T cells, methods for producing such cells, and preparation and medicament containing such cells and use of the cells.
  • CIITA is a transcription factor that activates the MHC II gene locus.
  • APC antigen presenting cell
  • Fig.1 The human MHC-II gene locus
  • Fig.2 The murine MHC-II gene locus
  • MHC class II Major histocompatibility complexes
  • T cell activation and antigen presentation depend on the amount of MHC-II on individual cells.
  • the regulation and expression of MHC-II genes is important for the control of immune responses.
  • the genes coding for the ⁇ and ⁇ chains of the HLA-DP, HLA-DQ and HLA-DR - class II molecules are located in the D region of the MHC locus Chromosome 6 grouped.
  • the genes are subject to a sometimes complex regulatory control. Their expression is generally coordinated and mainly restricted to cells of the immune system, such as B-lymphocytes, activated T-lymphocytes, macrophages (MQ), dendritic cells (DC) and certain specialized cells such as Kupffer cells and Langerhans cells.
  • B-lymphocytes B-lymphocytes, activated T-lymphocytes, macrophages (MQ), dendritic cells (DC) and certain specialized cells such as Kupffer cells and Langerhans cells.
  • MQ macrophages
  • DC dendritic cells
  • Kupffer cells and Langerhans cells.
  • lymphokines such as interferon-y or interleukin 4.
  • Class II molecules are also expressed transiently during the developmental pathway of many haematopoietic cell types.
  • MHC II genes represent a particularly complex type of regulated gene expression. This regulation affects not only the amount of expression of class II molecules but also the very restricted cell type specificity, since most cells in the body are normally MHC II negative.
  • the complexity of regulation includes two different types of control: constitutive expression in cells such as B cells and inducible expression in certain cell types such as monocytes and fibroblasts.
  • the family of genes includes the ⁇ and ⁇ chains from three different HLA class II isotypes. A number of protein factors can bind to the promoter of the MHC II genes in vitro and in vivo. CIITA is such a factor.
  • CIITA not only regulates the constitutive expression of MHC II genes in cells such as B lymphocytes, but also controls the inducible expression of the same genes in other cell types. It is therefore a factor that is involved in the general control of the MHC II genes.
  • CIITA is a protein with a sequence of 1130 amino acids in length, whose mRNA is only produced in small quantities. When CIITA cDNA is transfected into class II negative MHC cells, these cells begin to produce MHC II molecules 2 ', 3.4
  • the human gene for the MHC class II transactivator is on chromosome
  • mouse gene for CIITA is located on mouse chromosome 16.
  • CIITA is a non-DNA-binding coactivator that is recruited to the promoter via protein-protein interactions with the DNA-binding components of the enhososome.
  • CIITA activates transcription via an N-terminal transcription activation domain (AD).
  • AD N-terminal transcription activation domain
  • CIITA interacts directly with the RFX-ANK and RFX-5, subunits from RFX, as well as the B and C subunits from NF-Y and with CREB.
  • CIITA is a nuclear protein and contains three regions that appear important for transport to the cell nucleus. The first is located at the C-terminus of CIITA and contains five amino acids that resemble a nuclear localization signal (NLS).
  • the second region is a GTP-binding motif. Binding of GTP also appears to be necessary for the transport of CIITA into the cell nucleus.
  • the third region, called LRR, was also recognized by a mutation analysis as important for the direct transport into the cell nucleus. If CIITA does not get into the cell nucleus, the MHC II locus is not read either.
  • Fig. 3 Transcriptional regulation of the murine MHC-II.
  • the expression of CIITA is very strictly regulated.
  • the expression of CIITA dictates whether and to what extent MHC II genes are expressed.
  • the MHC II TA gene is therefore the main regulator of MHC II expression and therefore also has an essential immunomodulatory role.
  • Most cell types do not form CIITA and are consequently MHC II negative. Expression of CIITA and thus of MHC II can be activated by stimulating such cells with interferon- ⁇ (gamma).
  • fibroblasts include: fibroblasts, melanoma cells, macrophages, but also some primary cell types, such as mouse embryonic fibroblasts, peritoneal macrophages, microglia and astrocytes.
  • Transfection of MHC II negative cells with CIITA vectors is usually sufficient to induce MHC II expression.
  • a large (> 12 kb) and complex regulatory region contains various independent promoters that control the transcription of CIITA.
  • 4 promoters pl to pIV were identified in the human genes. Three of these p1, pH, pIII and pIV are also highly conserved in the mouse gene. The use of these promoters leads to the synthesis of distinct CIITA mRNAs (types I, III and IV). These contain alternative first exons that are spliced to a conserved second exon.
  • Analysis of the different MHC II-TA promoters has shown that their different activity is crucial for the complex expression pattern of MHC II genes. Each of them has a specific physiological relevance.
  • pl is very specific for dendritic cells, but pl is not the only promoter active in DCs. Significant transcriptions can also be detected by the plll promoter.
  • the type I transcripts contain an alternative first exon, which contains a translation initiation codon, which for a specific 94 amino acid long N-terminal extension of CIITA coded.
  • pIII is mainly used in B cells.
  • Type III-CIITA mRNA contains an alternative first exon that contains a translation initiation codon and encodes a specific 17 amino acid N-terminal extension.
  • pIV is activated by interferon gamma. Unlike Type I and Type III, Type IV mRNA does not contain a translation initiation codon and therefore does not code for an N-terminal extension.
  • the translation of CIITA is initiated by the first AUG of the second exon
  • a problem underlying the invention is to find therapeutically usable products for the reduction of pathological immune reactions, e.g. to provide for autoimmune diseases, transplants and allergies.
  • the problem is solved by the MHC II-producing non-antigen-producing cell (APC) according to the invention.
  • the invention relates to the use of non-antigen-presenting cells which are stimulated to produce MHC II for the ex vivo selection of T lymphocytes.
  • This can be achieved by interferon gamma or transfection with CIITA (CII transactivator).
  • Induction can be brought about by interferon gamma or the tract activator CIITA, which in turn can be achieved by introducing the gene for CIITA into the target cell.
  • the MHC II-producing cell according to the invention has the advantage that it cannot stimulate potent immune reactions via CD4 + T cells.
  • the switching off of T cells is improved by small amounts of CD80 (B7-1) and CD86 (B7-2), so that these molecules can also be produced in the MHC II-producing cell according to the invention.
  • B7-1 / B7-2 have a 20-50-fold higher affinity for CTLA-4 than for CD28. B7 in small amounts therefore preferentially binds to CTLA-4. In contrast to CD28, CTLA-4 has a tolerogenic effect on T cells. This effect is used.
  • the use according to the invention can be used in particular for the production of a medicament for suppressing immune reactions in the case of pathological Immune reactions, especially transplants, autoimmune reactions and allergies.
  • the non-AP cell can be selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchyma cells, chondrocytes and / or melanocytes.
  • Interferon gamma and / or CIITA can cause any cell to produce MHC II. Those that are easy to obtain are the best target cells for the desired treatment.
  • the invention also relates to a method for producing a composition which contains nAPC for a recipient, comprising the following steps:
  • the non-AP cells are incubated with interferon gamma and / or with CIITA and possibly with B7-1 and / or B7-2 and possibly with antigens,
  • the non-AP cell is typically selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchyma cells, chondrocytes, and / or melanocytes.
  • non-AP cells in particular the fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchyma cells, chondrocytes, and / or melanocytes.
  • a medicament can be produced from the composition according to the invention. This medicament is also the subject of the present invention.
  • the drug is not AP cells according to the invention. These cells can be infused into a patient.
  • the modified non-antigen presenting cells produce MHC II by incubation with interferon gamma (INF ⁇ ) or transfection with CIITA and they may also be transfected with B7-1 and / or B7-2.
  • the cells not presenting antigen can additionally be transfected with an antigen.
  • the infusion of the cells to be used according to the invention (modified non-AP) in the patient leads to an in vivo selection of T cells.
  • the cells not presenting antigen are selected or treated in such a way that they come into contact with T cells via MHC II, but cannot activate them.
  • the allogeneic non-AP cells in this case present the donor's alloantigens.
  • an alloreactive T cell encounters such a cell that does not present an antigen, it is permanently anergic (stopped), or sent to programmed cell death (apoptosis), or differentiated into a regulatory T cell.
  • a regu- latorial T cell is understood to be a T cell that suppresses immune responses to specific antigens, here alloantigens.
  • the non-AP cells that are syngeneic in this case present the allergens or autoantigens. If an allergy-triggering T cell encounters such a non-antigen presenting cell or an autoreactive T cell encounters such a non antigen presenting cell, it is permanently anergic (stopped) or sent to programmed cell death (apoptosis), or differentiated to the regulatory T cell.
  • a regulatory T cell is understood to be a T cell that suppresses immune words against specific antigens, here allergens or autoantigens.
  • a non-antigen-presenting cell which comes from the patient or, in the case of transplants, from the transplant donor and is characterized in that this cell is provided with CIITA or is caused to produce CIITA.
  • CIITA induces the synthesis of MHC II molecules, which in turn can present antigens in CD4 + T cells.
  • the T cell is not activated.
  • the T cells are switched off in this way and / or they are driven to programmed cell death (apoptosis). They can differentiate into regulatory T cells if the T cells recognize a presented antigen with their T cell receptor. With the at . Antigens recognized by this method are predominantly
  • Transplantation antigens [major histocompatibility complexes (HLA, MHC I, MHC II), rhesus factor, secondary histocompatibility antigens (mi- nor histocompatibility antige ⁇ s)], since these differ between donor and recipient of the transplant,
  • the cell is transfected with at least one gene for a CIITA.
  • the cells can be made to produce their MHC II molecules.
  • the cell to be used according to the invention can additionally or alternatively be transfected with CIITA as the protein.
  • CIITA protein the cells can be made to produce their MHC II molecules.
  • the cell to be used according to the invention can also be produced by incubation with INF ⁇ .
  • the cell according to A and / or D to be used according to the invention can additionally be transfected with B7-1 and / or B7-2 in order to force the negative selection of the T cells.
  • the cell according to A and / or D to be used according to the invention can additionally be transfected to allergens or autoantigens in order to force the negative selection of the T cells.
  • the cell according to the invention is infused into the patient.
  • they can be infused into lymph nodes.
  • they can be treated before the infusion in such a way that these cells will not proliferate. This is preferably achieved by irradiating the cells.
  • the methods that can be used make particular use of genetic engineering interventions on cells belonging to patients or transplant donors.
  • the interventions are performed using suitable probes and produce a protein that induces MHC II expression, preferably CIITA
  • a transfection of the non-antigen presenting cells causes an expression of CIITA and possibly additionally the production of B7-1 and / or B7-2 and possibly additionally the production of allergens or autoantigens. This is achieved in particular by nucleic acids which code for CIITA, B7-1 and B7-2 and allergens or autoantigens.
  • the nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides.
  • the DNA preferably contains regulatory elements such as enhancers, promoters, polyA-coding 3 ' ends for the transcription of the DNA in RNA.
  • the RNA in turn should contain regulatory elements for translating the RNA into protein.
  • the cells mentioned can be used ex vivo in a manner known per se by treatment with viruses, viral vectors, bacterial vectors, plasmids, by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into eukaryotic cells are transfected.
  • Said cell can be treated with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of CIITA and / or B7-1 and / or B7-2 and allergens or autoantigens are transfected, whereby T cells which have allogens, allergens or autoantigens, for example are presented on MHC molecules, bind to the cell according to the invention, are switched off.
  • CIITA CIITA, B7-1, B7-2, allergens, autoantigens and / or constituents (proteins, peptides, peptidomimetics) and / or combinations of these molecules can be used as molecules. These molecules hinder a stimulation and / or co-stimulation of T cells which take place in the presence of an allergy-triggering, allo- or autoantigen presentation and are brought into contact with the cell according to the invention.
  • the molecules can be generated by vehicles such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or others Techniques for introducing molecules into the cell according to the invention are transferred.
  • Nucleic acids can be transferred in particular by viruses, viral vectors, bacterial vectors, plasmids, which are transferred into the non-antigen-presenting cell by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules.
  • cells can be infused into the patient.
  • a medicament containing the preparation according to the invention is therefore also claimed according to the invention.
  • the medicament according to the invention is preferably formulated as an infusion solution for intravenous, injection into lymph nodes or intraperitoneal administration.
  • the formulation is chosen such that when the medicament is administered there is no significant impairment of the effectiveness of the cell presenting the antigen according to the invention.
  • Physiological saline is preferred as the infusion solution.
  • other solutions with a pH of 5.5 to 8.5 are also suitable.
  • Serum for example human serum, autologous or allologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable.
  • the non-antigen-presenting cell to be used according to the invention can be used according to the invention in particular for the production of a medicament for the treatment of immune reactions against allologic tissue features or allergens or autoantigens.
  • the rejection reactions to be treated are related to allologic tissue features, their gene sequences and / or partial sequences, in particular major histocompatibility complexes, MHC I, MHC II, rhesus factor, minor histocompatibility antigens (minor histocompatibility antigens).
  • the allergic or autoimmune reactions to be treated are particularly linked to allergens or autoantigens.
  • the cells not presenting antigen according to the invention can be infused into the patient.
  • Fig. 4 Cloned human CIITA in the pcDNA3.1 vector (see also the enclosed diagrams).
  • Fig. 5 shows FACS analysis that the primary human fibroblasts express MHCII after infection with hCIITA. The gene was transferred by lentiviral gene transfer.
  • Fig. 6 shows FACS analysis that the HEK293 cells express after transfection with hCIITA MHCII. It is a selected transgenic cell ink.
  • HEK293 cells human embryo kidney
  • plasmids which code for either CIITA or B7-2 or for both genes.
  • MLR mixed leukocyte reaction
  • CFSE-labeled human T cells from different donors were cultured for six days together with irradiated 293 cells. This results in an allogeneic system since the HEK293 cells carry different histocompatibility antigens (mainly MHC) than the T cells used.
  • MHC histocompatibility antigens
  • T cells need a primary stimulus above MHC II and B7 to proliferate.
  • the proliferation of the T cells was determined by FACS analysis of the decreasing CFSE staining during cell division; the total number of cells was calculated using Truecount Beads.
  • Fig. 8 shows a representative experiment.
  • Fig. 8 shows a T cell proliferation assay (human) with artificial APC (293 / MHCII-B7.2) and control cells (293, 293-B7-2 and 293-MHCII). 10 5 T cells were incubated with different amounts of artificial APC, control cells or alone for 6 days. The absolute number of proliferated CD4 and CD8 T cells was then determined.
  • 293 cells expressing both MHCII and B7.2 can stimulate the proliferation of naive CD4 T cells in a dose-dependent manner.
  • CD4 T cells cultured on 293 cells or 293 cells expressing either MHCII or B7-2 show no increased proliferation.
  • the proliferation of CD8 T cells is dose-dependent for all 293 clones used.
  • T cell proliferation was highest on the double positive 293 clones (MHCII and B7-2). This raised the question of whether T cells which are brought into contact with such artificial APC subsequently increase or decrease allogeneic immune reactions.
  • the T cells were separated from the 293 after the first incubation described above and brought together a second time with different 293 clones.
  • MACS-CD3 positive sorting 2. CFSE staining for 4 minutes, then wash twice with RF, then incubate in RF overnight, then plated in defined amounts in RF
  • FIG 11 shows CD4 T cell proliferation of T cells that have matured to double transgenic (MHC II / B7-2) or single positive (MHC II) 293. These T cells were incubated with various 293 clones. The ConA positive control was not shown for the 293-MHC II / B7-2, since this value (142962.9 +/- 11974.3) is far above the others.
  • FIG 12 shows CD8 T cell proliferation of T cells that have matured to double transgenic (MHC II / B7-2) or single positive (MHC II) 293. These T cells were incubated with various 293 clones. The ConA positive control was not shown for the 293-MHC II / B7-2, since this value (64307.9 +/- 367.7) is far above the others.
  • Fig. 13 Cloned murine CIITA in the pcDNA3.1 vector
  • Fig. 14 shows FACS analysis that the L929 cells express MHCII after transfection with mCIITA. It is a selected transgenic line.
  • proliferation assays should be carried out with non-APC and allogeneic T cells. It was found that 10% of the MACS-sorted T cells were contaminated with other cells, of which 3% were not B220-positive. Due to the processing and appearance in the FACS, it was probably macrophages and DC. In order to investigate the question of how such a contamination with donor DC affects the proliferation of the allogeneic T cells, these "impure" T cells were used in the assay. While we can assume that the T cells only proliferate after contact with MHCII and B7, i.e.
  • Fig. 16 Proliferated CD4 / CD4CD25 (A) and CD8 T cells (B) after first contact with L929. The individual L929 are shown. T cells without L929 (T cells only) served as a control of the
  • CD4 T cells the proliferation is strongest in the presence of the MHCII-positive L929.
  • Examination of another marker protein (CD25) showed that among the proliferated CD4 T cells were CD25 + cells when the L929 expressed MHCII.
  • the percentage of proliferated CD4 + CD25 + T cells were highest in the MHCII-B7-L929.
  • no "new" CD25 + T cells were produced.
  • interesting conclusions about the ratio of CD4 to CD8 T cells can also be drawn from this data. This is balanced in the MHCII-positive 5th L929, while in the MHCII-negative approaches the CD8 T cells preferentially proliferated.
  • the non-APC were able to generate regulatory T cells despite the presence of the DC donor. This effect is surprising since we expected that the non-APC are inferior to the donor DC and therefore do not affect the differentiation of the T cells. It should also be borne in mind that we only considered the "new", proliferated T cells, but not the previously existing CD25 + T cells, which make up up to 8% of the population. The function of the generated CD25 + T cells has yet to be explored. For this reason, we performed the proliferation assays described below when contacting this population 5 again and the corresponding controls with the allogeneic L929 cells.
  • T cells primed by non-APC should be checked for their proliferation / activation in a second MLR.
  • a reduced activation of L929-CIITA-B7.2, L929-CIITA primed T cells was expected upon contact with L929-CIITA and L929-CIITA-B7.2.
  • L929 and L929-B7.2 primed T cells were included as controls.
  • T cells Preparation of the T cells: 0
  • the T cells were prepared from Balb / c spleens 3 days before the initial incubation (primary MLR). After 3 days of cultivation, the T cells were control-stained for CD3-APC, CD4-PE, CD8-APC and PI.
  • Irradiated non-APC (L929-CIITA, L929-CIITA-B7.2, as controls L929 and L929-B7.2) were treated with Balb / cT cells for three days in the ratio shown in Table 1 in 12-well plates incubated.
  • T cells were carefully removed from the wells and transferred to 6-well plates. There they were cultivated for 2 days (rest phase).
  • non-APC primed T cells were stained with CFSE and again irradiated with non-APC (L929-CIITA, L929-CIITA-B7.2, as controls L929 and L929-B7.2) in the ratio shown in Table 2 in 48-well plates incubated.
  • T cells and non-APC were removed from the wells by pipetting and transferred to Eppis. The cells were then washed with PBA and then taken up in 50 ⁇ l PBA / sample. 0.5 ⁇ l CD4-PE, CD8-APC were added to this approach. Samples were stained on ice for about 30 minutes and then washed with PBA. TruCOUNT beads tubes (bead count: 50267) were filled with 2 ml PBA, vortexed and combined in a 50 ml falcon. The bead suspension was distributed to the samples to be measured with 500 ⁇ l per sample.
  • Fig. 18 Proliferated CD4 and CD8 T cells on second contact with allogeneic L929.
  • MACS-sorted Balb / c spleen T cells were mixed with artificial, allogeneic APC (L929-CIITA-B7.2, L929-CIITA, L929-B7.2 and L929) in a ratio of 1: 1 for Incubated for 3 days. After separation of the T cells from the non-APC and a 2-day rest phase, the now primed T cells were stained with CFSE and again combined with the various non-APC in a ratio of 1: 1 for 6 days. The T cell proliferation could be measured by diluting the CFSE with each cell division.
  • L929 and L929-B7.2 for example, proliferated CD4 + and CD8 + cells in contact with L929-CIITA-B7.2 twice as strongly as L929-CIITA or L929-CIITA-B7.2 pre-stimulated T cells (see Fig. 14) .
  • L929-CIITA corresponds to APC without Costimulus
  • L929-CIITA-B7.2 corresponds to immature DC
  • Anergic T cells would no longer proliferate and other regulatory allo- reactive T cells could shut down.
  • Fibroblasts are easily removed from the patient (e.g. from the skin)

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Abstract

L'invention concerne l'utilisation de cellules non présentatrices d'antigène qui sont transfectées avec le transactivateur de classe II (CIITA) pour la sélection in vivo de lymphocytes T.
PCT/EP2003/013402 2002-11-28 2003-11-28 Cellules non presentatrices d'antigene pour supprimer des reactions immunitaires pathologiques WO2004047860A2 (fr)

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AU2003289907A AU2003289907A1 (en) 2002-11-28 2003-11-28 Cells not presenting antigen for suppressing pathological immune reactions

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EP02026582.3 2002-11-28

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WO1998015626A2 (fr) * 1996-10-08 1998-04-16 Institute Of Child Health Molecule mutante ciita et ses utilisations
WO2001032189A1 (fr) * 1999-10-26 2001-05-10 Osiris Therapeutics, Inc. Surnageant issu de cellules souches mesenchymateuses pour la prevention et le traitement de reponses immunitaires lors de greffes
EP0648836B1 (fr) * 1993-08-26 2002-01-09 NovImmune SA Transactivateur du complexe majeur d'histomcompatibilité de la classe II et ses utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648836B1 (fr) * 1993-08-26 2002-01-09 NovImmune SA Transactivateur du complexe majeur d'histomcompatibilité de la classe II et ses utilisations
WO1998015626A2 (fr) * 1996-10-08 1998-04-16 Institute Of Child Health Molecule mutante ciita et ses utilisations
WO2001032189A1 (fr) * 1999-10-26 2001-05-10 Osiris Therapeutics, Inc. Surnageant issu de cellules souches mesenchymateuses pour la prevention et le traitement de reponses immunitaires lors de greffes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARMSTRONG T D ET AL: "Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, Bd. 94, Nr. 13, Juni 1997 (1997-06), Seiten 6886-6891, XP002224817 ISSN: 0027-8424 in der Anmeldung erw{hnt *
DATABASE EMBL 1. Oktober 1993 (1993-10-01), "MHC class II transactivator (CIITA)." XP002278858 gefunden im EBI Database accession no. P33076 *
STEIMLE V ET AL: "REGULATION OF MHC CLASS II EXPRESSION BY INTERFERON-GAMMA MEDIATED BY THE TRANSACTIVATOR GENE CIITA" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, Bd. 265, Nr. 5168, 1. Juli 1994 (1994-07-01), Seiten 106-109, XP002051563 ISSN: 0036-8075 *
YUN S ET AL: "Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain." TRANSPLANTATION. UNITED STATES 15 JUL 1998, Bd. 66, Nr. 1, 15. Juli 1998 (1998-07-15), Seiten 103-111, XP009010712 ISSN: 0041-1337 *

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AU2003289907A8 (en) 2004-06-18
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