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WO2003016905A2 - Methodes - Google Patents

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Publication number
WO2003016905A2
WO2003016905A2 PCT/GB2002/003753 GB0203753W WO03016905A2 WO 2003016905 A2 WO2003016905 A2 WO 2003016905A2 GB 0203753 W GB0203753 W GB 0203753W WO 03016905 A2 WO03016905 A2 WO 03016905A2
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mhc
peptide
hla
cell
peptide antigen
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PCT/GB2002/003753
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WO2003016905A3 (fr
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Bent Karsten Jakobsen
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Avidex Limited
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Priority to AU2002319547A priority Critical patent/AU2002319547A1/en
Publication of WO2003016905A2 publication Critical patent/WO2003016905A2/fr
Publication of WO2003016905A3 publication Critical patent/WO2003016905A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • the present invention relates to methods for assessing allo-specific T cell activity. Such methods find particular use in matching transplant and donor patients, as well as in monitoring alloreactive responses following a transplant operation.
  • Tissue-typing i.e., classification of a patient's Human Leukocyte Antigen (HLA) type
  • HLA Human Leukocyte Antigen
  • graft rejection owing to alloreactivity can be controlled by powerful but dangerous immunosuppressive drugs. Because the risk of rejection is greatest in the early post-transplant period, maximum immunosuppression is given at this time. Protocols vary, but most regimens incorporate high-dose steroids, high-dose calcineurin inhibitors (cyclosporin or tacrolimus) and an anti-proliferative agent (azathioprine or mycophenolate mofetil). Anti-T cell antibodies are also used in some situations. The most widely used preparations are Muromonab CD3 (OKT3) and anti- thymocyte globulin/anti-lymphocyte globulin (ATG/ALG).
  • Maintenance therapy usually consists of the same three classes of drugs used at lower doses: a calcineurin inhibitor, an anti-proliferative agent and glucocorticoids.
  • a calcineurin inhibitor used at lower doses: a calcineurin inhibitor, an anti-proliferative agent and glucocorticoids.
  • a number of new drugs are undergoing clinical evaluation, but equally important is the determination of the safest and most effective combinations of existing drugs.
  • the relatively wide choice of potent immunosuppressive drugs allows flexibility in minimising side effects for the individual patient, but the side effects of long-term immunosuppression still remain a challenging clinical problem.
  • HLA-A the weakest
  • HLA-B the most potent
  • HLA-DR the most potent
  • HLA typing was performed with a panel of antisera specific for different antigenic products of the system in an assay that became known as the complement-dependent cytotoxicity (CDC) test (Mittal, et al, Transplantation (1968) 6: 913-27), or serology. CDC is robust and can be performed in three hours. However, there are drawbacks: viable lymphocytes are required, the antibodies required are generally non-renewable and the technique has limited powers of resolution, particularly for Class II HLA. Despite the lack of standardisation and simplicity, the CDC crossmatch assay remains a prerequisite in kidney transplantation.
  • CDC complement-dependent cytotoxicity
  • PCR The development of PCR allowed the evolution of improved molecular HLA-typing techniques and extensive sequencing of HLA alleles followed. Transplantation laboratories were able to use PCR to amplify the polymorphic regions of HLA genes that could subsequently be analysed for the polymorphism within the amplicon, thus establishing tissue-type. Methods can be tailored to identify broad specificities (low resolution), serological specificities (medium resolution) or to discriminate between more than 90% of the alleles in the loci analysed (high resolution). In solid organ transplantation, medium resolution typing is usually carried out, whereas in unrelated bone marrow transplantation, low or medium resolution typing is always followed up with high resolution typing.
  • probe hybridisation Techniques for analysing the polymorphisms in amplified DNA can be divided into two basic groups: probe hybridisation and direct amplicon analysis.
  • Current probe- based techniques generally require lengthy post-PCR steps which mean that the fastest method cannot be completed in less than five hours. However, there are other techniques which are suitable for limited numbers of samples that can be carried out more quickly. These include PCR-RFLP, PCR using sequence-specific primers, heteroduplex analysis and other conformational assays (Bunce, et al, Transplantation (1997) 64: 1505-13) .
  • HLA-matched renal allografts have a superior graft survival compared with HLA-mismatched transplants.
  • an increasing number of studies indicate that not every HLA mismatch has the same detrimental effect on survival.
  • HLA-DR mismatches seem to have a strong influence on survival, especially in the first six months after transplantation; this is not surprising as the HLA-DR molecules are mainly expressed on dendritic cells in the transplanted organ. The donor dendritic cells are eventually replaced by recipient-derived cells.
  • HLA-B mismatches have a more profound effect on renal allograft survival than HLA-A mismatches.
  • differential effects of mismatches on graft survival have been reported at the level of individual alleles witiiin a locus. Maruya et al (Maraya, et al, In Clinical Transplants 1993 (eds PI Terasaki and JM Cecka), (1994) p511. UCLA Tissue Typing Laboratory, Los Angeles) were able to define a number of specific HLA mismatches that gave a graft survival similar to HLA-identical combinations. These mismatches have been called "permissible mismatches".
  • HLA matching policies are based on the selection of recipients who have the same HLA specificities as the donor; this is called "structural matching".
  • structural matching The advent of molecular typing techniques (see above) has led to an enormous increase in the identification of HLA polymorphisms and clearly shows that structural matching of unrelated donor and recipient organs and tissues is practically unachievable. It is also clear that HLA matching policies would be more effective if they involved "functional matching" where the HLA molecules of the donor should be non-stimulatory to the immune system of the recipient.
  • Reliable in vitro assays that mimic allograft rejection are essential to test whether the HLA profile of a particular donor is acceptable to the immune repertoire of the recipient.
  • Limiting dilution assays have been used to monitor the frequency of alloreactive T lymphocytes (Deacock, et al, J Immunol Methods (1992) 147: 83-92; Sharrock, et al, Immunol Today (1990) 11: 281-6).
  • the avidity of the T cells recognising donor antigen also needs to be taken into consideration; the presence of CTL that do not require CD8 molecules to stabilise then- binding (i.e., high avidity) is significantly associated with graft rejection (Roelen, et. al, Transplantation (1995) 59: 1039-42). In contrast, low avidity CTL are found mainly amongst graft-infiltrating cells in the absence of rejection.
  • Primed CTL require re-stimulation with donor antigen in vitro before they express their cytolytic function whereas primed CTL are already cytotoxic for donor cells without re-stimulation in vitro.
  • a significant association has been found between the presence of primed CTL and graft rejection (Naessen, et al, Clin Exp Immunol (1992) 88: 213-9).
  • Epstein Barr virus is associated with post- transplant lymphoproliferative disease (PTLD) in immunocompromised patients.
  • PTLD is a spectrum of mainly B cell diseases that range from polyclonal lymphoproliferative disorders which resolve when immunosuppression is halted to highly malignant lymphomas. It is believed that the underlying immunosuppression inhibits the virus-specific CTL response that would normally control viral replication and maintain latency (Khatri, et al, J Immunol (1999) 163: 500-6).
  • EBN matching between recipient and donor is unclear, although it has been suggested that PTLD does not occur with any greater frequency in mismatched patients (Harwood, et al, Pediatr Transplant (1999) 3: 100-3).
  • Early diagnosis of EBN- associated PTLD is important because many patients respond to a reduction in immunosuppression, and several groups are developing assays to detect EBN markers (e.g., D ⁇ A) in serum.
  • EBN markers e.g., D ⁇ A
  • the development of an assay to monitor the risk of graft rejection through an alloreactive response and therefore reduce the need for immunosuppression may also help to reduce the incidence of EBN-associated PTLD.
  • CMV herpes virus cytomegalovirus
  • a diagnostic feature of acute rejection is infiltration of the allograft parenchyma by lymphocytes, a process regulated by the induction of adhesion molecules on vascular endothelial cells and their ligands on leucocytes.
  • CMN is also associated with an increased expression of MHC class II on multiple cell types. An upregulation of these molecules could also contribute to graft failure.
  • a diagnostic tool for detecting the vigour of the immune response between a recipient and potential donors may predict the likelihood of rejection in CMN positive recipients.
  • HLA typing has provided a major breakthrough in assessing how to match donor and host so as to reduce the risk of immune cross-reactivity.
  • perfect HLA matching is virtually impossible to achieve.
  • HLA typing only provides an indirect prediction of the likely level of crossreactivity that can be expected between host and transplant tissue.
  • the critical issue is how vigorous the immune responses to the particular non-matched HLA types will be. Immune responses to different HLA types could vary substantially, and therefore a high degree of variability in transplant rejection is observed even when HLA matching is applied.
  • T cells and their T cell receptors are normally restricted by self MHC molecules and will only respond when presented with specific peptides.
  • substantial effort has gone into developing methods that allow the identification or targeting of the subset of T cells that are specific for a particular peptide antigen.
  • the present inventors have provided a method that allows the identification or targeting of T cells which are specific for particular MHC (in humans HLA) and not for a particular peptide antigen. This approach has particular use in detecting or targeting T cells that display alloreactivity as is observed in the rejection of tissue and organ transplants.
  • a method for determining whether a T cell reacts with a predetermined Major Histocompatibility Complex (MHC) type comprising: bringing a sample comprising said T cell into contact with a plurality of molecules of said MHC type, each MHC molecule being complexed with a peptide antigen whose contribution to a T cell receptor binding the MHC-peptide antigen complex is minimised; and determining whether said plurality of MHC molecules causes activation of the T cell.
  • MHC Major Histocompatibility Complex
  • the method of the present invention can be used to detect an alloreactive T cell restricted by the particular MHC type. Such a T cell may then form the basis of a therapy against the alloreaction.
  • the method of the present invention finds particular use in determining the reactivity of an individual towards each of a plurality of Major Histocompatibility Complex (MHC) types (i.e. the ability of the individual to stimulate a T cell reaction against a particular MHC).
  • MHC Major Histocompatibility Complex
  • the method of the present invention has particular utility in matching transplant and donor patients, as well as monitoring alloreactive responses following a transplant operation.
  • the method of the present invention is preferably used to determine the reactivity of a human individual.
  • the MHC types are preferably Human Leukocyte Antigen (HLA) types. Because in most - but not all - instances, the method of the present invention is for use in determining the reactivity of a human, for example in an organ transplant procedure, for convenience, reference will be made in the following to HLA. However, it is to be understood that such references are intended to include MHC types of non-human animals.
  • HLA Human Leukocyte Antigen
  • the method of the present invention provides a convenient method for directly assessing, from patient T cell sample, e.g. a small blood sample, the relative levels of immune cross-reactivity towards a range of different HLA types.
  • patient T cell sample e.g. a small blood sample
  • the ability to obtain a direct assessment of functional donor and host immune reactivity towards a range of HLA types constitutes a fundamental improvement over the simple genetic HLA matching of the prior art. With such information available, it is possible to combine donor and host so as to minimise the actual immune cross- reactivity, rather than merely minimising mis-matched genotypes. In turn, this can lead to a reduced requirement for treatment with immunosuppressive drugs following the transplant operation, thereby reducing the often severe side-effects associated with this type of therapy.
  • the assay can be performed again at regular intervals, or when required, in order to follow the development in allo-specific T cell activity directed against the transplanted tissue.
  • the assay can be used as an indicator for decisions on the level of immuno- suppressor therapy to be employed at this stage in order to avoid rejection.
  • the method of the present invention finds use in matching a transplant patient and a xeno-transplant organ, and for monitoring the progress of such a transplant.
  • T cell alloreactivity is less dependent on antigen peptide than is the case for conventional, antigen-primed, and self-HLA type restricted, immune responses.
  • activation of alloreactive T cell responses is therefore more dependent on interactions between T cell receptors (TCRs) and the HLA heavy chain component of the antigen complex, than on TCR-peptide interactions.
  • T cells mature in the thymus where they undergo at least two selection mechanisms, generally referred to as positive and negative selection.
  • the structures of most, or all, TCRs are believed to share certain general architectural features (Chothia, et al, Embo J (1988) 7: 3745-55) that provide a framework suitable for MHC/peptide binding by the variable complementarity determining regions (CDRs).
  • CDRs variable complementarity determining regions
  • T cells with high affimty for one of the self MHC molecules will be negatively selected (Amsen & Kruisbeek. (1998). Immunol Rev 165: 209-29. Sebzda, et al (1999). Annu Rev Immunol 17: 829-74). It is unclear to what extent the peptide ligands presented influence these selection procedures.
  • Mature circulating T cells are believed to be able to recognise a substantial number of different peptide antigens presented by the same MHC molecule.
  • a recent review (Mason & Powrie, Curr Opin Immunol (1998) 10: 649-55) estimates that an individual TCR may be able to bind up to IO 6 different peptides.
  • each MHC molecule has an estimated capacity to bind ⁇ 10 10 different peptides
  • each T cell will still be highly selective, responding only to 0.01% of the possible peptide antigens presented by the particular MHC molecule.
  • the promiscuity displayed by T cells appears to be even more pronounced when exposed to cells presenting HLA molecules expressed from different alleles than those on which they were selected in the thymus.
  • T cells exposed to cells expressing foreign MHC alleles will display "allo-reactivity", a phenomenon that is usually observed when an organ or tissue is transplanted into a heterologous host.
  • the T cell alloreactivity is usually vigorous, leading to destruction (rejection) of the foreign cells.
  • allo-reactive T cells may have TCRs with high affimty for the allo-restrictive MHC molecule.
  • EBN Epstein-Barr vims
  • HLA-B44 negative have T cell alloresponses to HLA-B44 that coincide with their HLA-B8/EBN responses. In most patients, this is mediated by an immuno-dominant, "public", T cell clone (Burrows, et al, JExp Med (1994) 179: 1155-61). However, this clone is not observed in patients that are HLA-B44 positive, indicating that, in these patients, the public T cell clone is eliminated by negative selection. Instead, HLA-B44 positive patients display a variety of T cell responses to HLA-B8 presenting EBN antigen (Burrows, et al, EurJ Immunol (1997) 27: 1726-36).
  • peptide antigens mediating allo-reactivity are relatively easy to identify.
  • a T cell clone specific for EBN antigen restricted by HLA-B8 was found to be alloreactive for HLA-B35.
  • Screening of the human protein sequence database (SwissProt) was used in a study to search for putative peptide sequences that would act as antigens when presented by HLA-B35.
  • a previously defined fine specificity of the T cell clone for peptides presented by HLA-B8 was used to search for peptides with suitable HLA-B35 anchor residues. Strikingly, of 37 peptides tested, two acted as strong antigens for the T cell clone when presented by HLA-B35 (Burrows, et al, Eur J Immunol (1997) 27: 1726-36).
  • the method of the present invention is intended to optimise the detection of T cells with the ability to recognise certain HLA types.
  • the contribution of the peptide antigen to a T cell receptor binding the MHC-peptide antigen complex may be minimised by complexing the MHC molecules with one of (a) a peptide antigen which presents substantially no T cell recognition features, and (b) a peptide antigen in which one or more T cell recognition features are randomly present.
  • the peptide antigens in the HLA molecules used in the method of the present invention are designed not to contribute to the T cell binding the HLA.
  • the different types of HLA/peptide complexes may be provided in which the "normal" antigenic peptides have been substituted for "null" peptides which provide the absolute minimum of features for TCR recognition.
  • Such peptides preferably comprise alanine residues except at amino acid positions which are required to bind the peptide to the HLA heavy chain (so-called “anchor residues” ).
  • the null peptides can therefore form a complex with the HLA, but otherwise the peptide has no amino acid side chains (other than hydrogen) which could contribute to T cell binding.
  • null peptides may have glycine and/or serine residues to provide the minimum of T cell recognition features.
  • the "normal" peptides can be substituted for peptides from designed peptide libraries in which certain positions are randomised so as to make it more likely that antigen complexes are included that stimulate a larger proportion of T cells. Because of the high degree of variation in these peptide antigens, T cell recognition will not be dominated by a subset of the T cells which are specific for one particular antigen. Instead, the observed T cell response will be dominated by the bias towards the particular HLA molecule presenting the redundant antigens. Where only one or a few positions are randomised in the peptide antigen, the T cell response will be dominated by those cells which can react to the HLA molecule supported by minimal features by the peptide.
  • the T cell response will be dominated by those cells which react to the HLA molecule supported by a variety of features supplied by the peptides, again biasing the response in favour of those T cells that rely mainly on contacts to the HLA heavy chain for activation.
  • each of the plurality of HLA molecules may be provided in an individual reaction chamber, e.g.
  • the sample of the individual's T cells may be provided by a sample of peripheral blood leucocytes (PBLs) such that the method of the present invention detects all those T cells within the sample which are alloreactive to the provided HLA molecules.
  • PBLs peripheral blood leucocytes
  • HLA Major Histocompatibility Complex
  • HLA molecule as a soluble complexes exists or can be developed.
  • the haploid genome contains three loci coding for type A, type B, and type C respectively of HLA class I molecules. A large number of alleles have been identified for each locus and any individual will express three different HLAs per haploid content equalling up to six different molecules on each cell.
  • HLA-A2 describes the HLA-A molecules sharing the A2-characteristic serologic determinants (f. ex. HLA-A0201 - HLA-A02012).
  • HLA-A* A0201 a number is assigned to the alleles of the loci (f.ex. HLA-A* A0201) as their sequences become known.
  • the internet provides some helpful sites that incorporate the HLAs as their sequences appear, along with information regarding their distribution in the population.
  • the IMGT/HLA Database fl ⁇ ttp://www.ebi.ac.uk/imgt/hla is part of the international ImMunoGeneTics (EVIGT) project and provides a specialist sequence databases for sequences of the human major histocompatibiUty complex (HLA). This includes all official sequences for the WHO HLA Nomenclature Committee For Factors of the HLA System. Development of this database has been undertaken by James Robinson, Julia G. Bodmer and Steven G.E. Marsh. Two sites providing programmes for predicting peptide motifs in any given protein, authentic or synthetic have been opened. Both sites are supervised by acknowledged experts. In Germany, the
  • SYFPEITHI-site is provided by Dr. Hans-Georg Rammensee's group (Hans-Georg Rammensee, Jutta Bachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An Internet Database for MHC Ligands and Peptide Motifs (access via : http://www.uni-tuebingen.de/uni/kxi ). This site is based on published reviews (Rammensee, et al, Immunogenetics, 1995, 41:178-228; Rammensee, et al: MHC ligands and peptide motifs. Austin Bioscience 1997).
  • the Web site was created by Ronald Taylor of the Bioinformatics and Molecular Analysis Section (BIMAS), Computational Bioscience and Engineering Laboratory (CBEL), Division of Computer Research & Technology (CIT), National Institutes of Health, in collaboration with Dr. Parker. Such sites allow peptide antigens to be designed which have the required “anchor” residues, but which otherwise are either "null” peptides or are randomised.
  • Table I lists known HLA class I molecules, and is compiled from the "BHVIAS-site". Included is a list of semi-optimised synthetic poly-alanine peptide sequences calculated on the basis of the "HLA Coefficient Tables" used by the peptide prediction programme (Peptides marked by ⁇ were designed on the basis of information in the indicated literature since no HLA Coefficient tables were available for these HLA types) and the references cited by the BIMAS-site for each HLA molecule.
  • HLA-A*0205 del Guercio, et al. J Immunol. 1995 154 (2): 685-93.
  • the LMGT/HLA database is more useful.
  • the database contains information regarding the amino acid and DNA sequences, ethnic distribution, information about the cell line from which the sequence was isolated, links to the databases (Genbank/EMBL/SwissProt) harbouring the original information, and medline links to the relevant literature of the particular entry.
  • a query in the keywords field of this database using the search parameter "class II" returned 472 entries most representing an individual allele of a classical or non- classical HLA class II molecule.
  • Soluble class I MHC-peptide complexes were first obtained by cleaving the molecules of the surface of antigen presenting cells with papain (Bjorkman, et al, J Mol Biol (1985) 186: 205-10). Although this approach provided material for crystallisation, in recent years it has been replaced for class I molecules by individual expression of heavy and light chains in E. coli, followed by refolding in the presence of synthetic peptide (Gao, et al, Prot. Sci.
  • Full length DRB 1*0401 has been expressed on the surface of Drosophila melanogaster Schneider 2 cells under control of a copper sulphate-inducible Drosophila metallothionein promoter (Hansen, et al, Tissue Antigens (1998) 51: 119-28).
  • This approach is readily modified to produce soluble MHC class II molecules simply by expressing a truncated version of the protein which contains a biotinylation tag sequence in place of the transmembrane domain. This protein will be secreted in a soluble form instead of bound to the extracellular surface of the cell membrane.
  • HLA-DR1 The ⁇ - and ⁇ -chains of HLA-DR1 have been expressed in E.coli as inclusion bodies and purified separately under denaturing conditions prior to co-refolding in vitro (Frayser, et al, Protein Expr Purif (1999) 15: 105-14). The synthesised protein was soluble, bound peptide in the expected manner, and was stable.
  • the MHC types are provided as multivalent complexes.
  • Such complexes may be in the form of monomers of the MHC coated in close proximity on a surface, e.g. provided in a liposome or attached to a surface, either directly or via biotin to avidin which is attached to the surface or alternatively in the form of MHC multimers.
  • a suitable surface is the multiscreen Immobilon-P (Millipore) 96 well filtration plate which contains a sterile, high protein binding hydrophobic Immobilon-P membrane.
  • the tetrameric peptide-MHC complex may be made with synthetic peptide, ⁇ 2microglobulin (usually expressed in E.coli), and soluble MHC heavy chain (also expressed in E. coli).
  • the transmembrane domain is truncated from the heavy chain and replaced with a protein tag constituting a recognition sequence for the bacterial enzyme BirA (Barker & Campbell, JMol Biol (1981) 146: 451-67; Barker & Campbell, J Mol Biol (1981) 146: 469-92; SchsAz, Biotechnology N Y (1993) 11: 1138-43).
  • Bir A catalyses the biotinylation of a lysine residue in a somewhat redundant recognition sequence (Schatz, Biotechnology N Y (1993) 11: 1138-43) .
  • the specificity is high enough to ensure that the vast majority of protein will be biotinylated only on the specific position on the tag.
  • the biotinylated protein can then be covalently linked to streptavidin which has four binding sites resulting in a tetrameric molecule of peptide-MHC complexes (Airman, et al, Science (1996) 274:94-6).
  • Multimeric class II MHC/peptide complexes have also been described (Crawford, et al, Immunity (1998) 8: 675-82), and it is becoming well established that both class I and class II restricted T cells can be activated by soluble MHC complexes provided in multimeric formulations.
  • MHC/peptide tetramers are now being used for the detection and quantification of antigen-specific T cells, and the technology holds promise for a multitude of uses in diagnostics and for the development of novel antigen-specific therapies (McMichael & O'Callaghan, J Exp Med (199 ) 187: 1367-71).
  • the method of the present invention is intended to detect T cells which recognise certain MHC types regardless of the peptide.
  • the peptide antigens in the MHC molecules used in the method of the present invention are peptides which are designed not to contribute to the T cell binding the MHC.
  • the peptides may be "null" peptides that provide a mimmum of features for TCR recognition.
  • the MHC complexes may contain designed peptide sequences in which one or a few positions are randomised in order to provide minimal features for TCR recognition.
  • T cell ELISPOT assay The principle of this assay is that activation of signal transduction induces ⁇ -interferon production and secretion by T cells. By plating the T cells, and incubating with an appropriate antibody; the number of activated cells can be easily counted. Thus, the counts obtained from each plate provide a measure of T cell reactivity to the HLA type molecule used for incubation. By comparing the activated T cell counts obtained by incubation with the different alloreactive and self-specific HLA molecules, a relative measure of crossreactivity is obtained.
  • WO98/23960 describes a method for detecting activation of T cells.
  • Enzyme-linked immunospot (ELISPOT) assays have been used to determine the frequencies of cytokine-secreting T lymphocytes in peripheral blood from patients with viral, cancerous, and infectious diseases (Hagiwara, et al, Cytokine (1995) 7: 815- 22; Herr, et al, J Immunol Methods (1996) 191: 131-42; Lalvani, et al, JExp Med
  • the ELISPOT assay detects secreted cytokine molecules (IFN- ⁇ , IL-2, IL-4, IL-6, LL-10, TNF- ⁇ ) in the immediate vicinity of the cell from which they are derived, and release of cytokine(s) represents a measure of effector function.
  • cytokine molecules IFN- ⁇ , IL-2, IL-4, IL-6, LL-10, TNF- ⁇
  • Each spot in the read-out represents a "footprint" of the original activated T lymphocyte in response to antigenic peptide. Therefore, the numbers of spots provide an indication of the frequency of T cells that respond to the antigen which is used in the assay.
  • LDA Limiting-dilution analysis
  • ELISPOT assay for quantification of the frequency of antigen specific T cell reactivity in peripheral blood lymphocytes.
  • An ELISPOT assay was used for the detection and quantification of CD8 T lymphocytes isolated from peripheral blood, recognising melanoma peptide antigens presented by HLA- A*0201.
  • CD8 T lymphocytes were isolated from peripheral blood and stimulated for 40 h with HLA-A2*0201 positive T2 cells loaded with melanoma peptide.
  • Tumour necrosis factor ⁇ (TNF- ⁇ ) secreted by activated CD8 + T lymphocytes in response to melanoma peptide was trapped on nitrocellulose membranes precoated with anti-TNF- ⁇ antibodies and was then immunochemically visualised as spots (Herr, et al, J Immunol Methods (1996) 191: 131-42). Another study described the use of the ELISPOT assay to detect CD8 + T lymphocytes specific for influenza virus antigenic peptides.
  • PBMC Peripheral blood mononuclear cells
  • BCLs result in strong EBV-specific responses. Allo-specific and EBV-specific responses can be circumvented by using the autologous fresh PBMCs themselves to present the antigenic peptide.
  • Peptide-specific CD8 + T lymphocytes displayed IFN- ⁇ release within 6 hour of peptide antigen stimulation and the frequency of peptide- specific CD8 + T lymphocytes enumerated by the ELISPOT assay was higher than the corresponding CTL precursor frequency as detected by LDA.
  • the ELISPOT assay appears to be a more sensitive assay for detecting peptide-specific CD8 + T lymphocytes in peripheral blood.
  • the soluble HLA complexes will be generated with either 'null' peptides or peptides containing a degree of randomness at selected amino acid, positions. Both of these types of complexes can be generated with synthesised peptide or with the peptide expressed as a fusion with ⁇ 2microglobulin. In the former case, for class I HLA molecules, the heavy chain, the light chain (i.e. ⁇ 2m) and the peptide are folded together. For class II HLA molecules, the complex is expressed 'empty' and loaded with peptide subsequently.
  • the invention provides a cloning vector, which is preferably DNA, encoding an MHC subunit (preferably human) into which a nucleotide (e.g.synthetic DNA) encoding a peptide antigen sequence of interest can be inserted such that expression of the vector produces a fusion protein comprising the MHC subunit with the peptide antigen fused thereto via a linker sequence.
  • a nucleotide e.g.synthetic DNA
  • the subunit is preferably ⁇ 2microglobulin, with the peptide antigen fused at the N- terminus thereof.
  • This vector may have different selective markers for propagation in E.coli,' different restriction enzyme sites for cloning; a different length or composition of the linker sequence between the antigenic peptide sequence and ⁇ 2microglobulin; or with altered codon usage for expression of ⁇ 2microglobulin.
  • the peptide sequence to be expressed as a fusion with ⁇ 2microglobulin can be altered by the polymerase chain reaction (PCR) or by cloning in synthetic DNA oligomers corresponding to the desired peptide sequence.
  • recognition motifs for the restriction enzymes Ndel and BamHI in the DNA sequence facilitate alteration of the peptide sequence which is expressed together with ⁇ 2microglobulin.
  • the subunit may be the ⁇ - or the ⁇ -chain, with the peptide antigen fused at the amino terminus thereof.
  • the invention also provides, in a third aspect, a cell transformed with such a vector.
  • ⁇ 2microglobulin can be expressed in a number of factory cell lines, e.g., E.coli, Pichia Pastoris and mammalian cell lines. Similarly, peptide- ⁇ 2microglobulin fusion proteins may be expressed in any of these systems. E.coli may be the most advantageous because it is less likely that protease processing in this organism will remove the fused peptide from the ⁇ 2microglobulin polypeptide.
  • the invention provides a multivalent class I or class U MHC/peptide complex, preferably a multimer, in which the peptide is optimised for assessment of T cell responses that are predominantly mediated through TCR-MHC contacts rather than through TCR-peptide contacts.
  • the peptide may be a 'null' peptide which presents substantially no T cell recognition features .
  • all amino acids in the peptide, except those residues that bind the peptide in the MHC groove (the 'anchor' residues) may be alanine, glycine and/or serine (such as glycine-serine mixtures) residues.
  • the peptide may be one in which T cell recognition features are randomly present.
  • the peptide may be from a library of peptides in which certain positions are a mixture of selected or random residues.
  • the other positions in these peptide libraries would be occupied by neutral residues, typically alanine, except for the 'anchor' residues which would be chosen as optimal as possible for binding to the respective HLA type molecules.
  • the randomised positions are for providing a small peptide contribution to TCR binding to the HLA complex, so that binding is being dominated by TCR-heavy chain contacts.
  • the antigen peptide may be covalently linked to one of the subunits in the MHC complex by constituting part of the same polypeptide chain, and preferably forms part of a fusion protein with one of the MHC subunits.
  • the peptide may be covalently linked to ⁇ 2-microglobulin via a flexible linker sequence.
  • the peptide may be covalently linked to the amino terminus of the ⁇ or ⁇ chain thereof via a flexible linker sequence (Kozono, et al, Nature (1994) 369: 151-4).
  • the invention also provides, in a fifth aspect, a kit for determining whether a T cell reacts with a predetermined Major Histocompatibility Complex (MHC) type, the kit comprising: a plurality of molecules of said MHC type, each MHC molecule being complexed with a peptide antigen whose contribution to a T cell receptor binding the MHC-peptide antigen complex is minimised.
  • MHC Major Histocompatibility Complex
  • the kit is for determining the reactivity of an individual towards each of a plurality of Major Histocompatibility Complex (MHC) types, and comprises a plurality of molecules of said plurality MHC types.
  • MHC Major Histocompatibility Complex
  • the kit preferably further comprises means for determining whether any of said MHC types causes activation of the individual's T cells.
  • means for determining whether any of said MHC types causes activation of the individual's T cells may be the reagents required for an enzyme-linked immunospot assay (ELISPOT).
  • ELISPOT enzyme-linked immunospot assay
  • the kit may further comprise instructions allowing a user of the kit to practice the invention.
  • Figure 1 shows the DNA sequence of a plasmid, pBJ196 which can be used for expression of a polypeptide which is a fusion of three elements: a peptide corresponding to an antigenic peptide from influenza virus ('flu matrix peptide) which is known to be presented by HLA-A0201 , a flexible linker sequence mainly comprised of Glycine and Serine residues, and ⁇ 2microglobulin. The amino acid sequence of the resulting fusion protein is also shown.
  • Figure 2 shows the DNA and amino acid sequences of the N-terminal part of the peptide-linker- ⁇ 2microglobulin fusion polypeptide produced by plasmid pEX013, a modification of pBJ196.
  • Figure 3 shows the DNA and protein coding sequences of the N-terminal part of the peptide-linker- ⁇ 2microglobulin fusion polypeptide produced by plasmid pEX014, a modification of pBJ196.
  • Example 1 A DNA plasmid vector for expression of peptide- ⁇ lmicroglobulin fusions in E.coli.
  • This example describes the design and construction of a DNA plasmid vector for expression of peptide- ⁇ 2microglobulin fusions in E.coli.
  • the vector was designed so that the peptide to be expressed can be easily changed by inserting a pair of synthetic DNA oligonucleotides encoding the new peptide sequence.
  • a PCR reaction was performed with cDNA generated from human cytotoxic T lymphocytes (CTL) as a template.
  • CTL cytotoxic T lymphocytes
  • the PCR reaction generated a DNA fragment comprising the sequence encoding human ⁇ 2microglobulin as expressed on the cell surface, i.e. without the signal peptide.
  • the fragment also contained sequence 5' of the ⁇ 2microglobulin gene which encodes a linker fusion sequence.
  • the fragment was cloned into the DNA expression vector pGMT7 (Studier, et al, Methods Enzymol (1990) 185: 60-89 Issn: 0076-6879) using the unique BamHI (GGA TCC and HindUI (GAAJTC) restriction sites which are underlined in the primer sequences above.
  • the resulting DNA plasmid was then modified by cloning a pair of synthetic DNA oligonucleotides into the unique Ndel and BamHI sites.
  • the sequences of the DNA oligonucleotides were as follows:
  • the resulting DNA plasmid, pB Jl 96 can be used for expression of a polypeptide which is a fusion of three elements: a peptide corresponding to an antigenic peptide from influenza vims ('flu matrix peptide) which is known to be presented by HLA- A0201, a flexible linker sequence mainly comprised of Glycine and Serine residues, and ⁇ 2microglobulin.
  • the DNA sequence of plasmid pBJ196 is shown in Figure 1. The DNA sequence of pBJ196 was verified by dideoxy sequencing. The amino acid sequence of the fusion protein is indicated above the DNA sequence of the relevant region.
  • the vector pBJ196 contains a T7 promoter for high-level expression in a suitable E.coli strain such as BL21DE3pLysS or BL21DE3pLysE
  • Example 2 DNA plasmid vector for expression of an HLA-A2 nullpeptide- ⁇ 2microglobulin fusion in E.coli.
  • This example describes the design and construction f a DNA plasmid vector for expression of a HLA-A2 null peptide- ⁇ 2microglobulin fusion.
  • Nector pBJ196 was modified by restriction enzyme digestion with enzymes ⁇ del and BamHI and insertion of the synthetic oligonucleotide pair:
  • FIG. 2 shows the D ⁇ A and protein coding sequences of pEX013 covering the ⁇ -terminal part of the peptide-linker- ⁇ 2microglobulin fusion polypeptide. This sequence was verified by D ⁇ A dideoxy sequencing.
  • the peptide fused to ⁇ 2microglobulin in the protein expressed from pEX013, ALAAAAAAN, consists of Alanine residues in all positions except positions 2 and 9 which are optimised for anchoring the peptide to the HLA-A*0201 heavy chain.
  • Example 3 A DNA plasmid vector for expression of an HLA-B8 nullpeptide- ⁇ 2microglobulin fusion in E.coli.
  • Nector pBJ196 was modified by restriction enzyme digestion with enzymes ⁇ del and BamHI and insertion of the synthetic oligonucleotide pair:
  • FIG. 3 shows the D ⁇ A and protein coding sequences of pEXO 14 covering the ⁇ -terminal part of the peptide-linker- ⁇ 2microglobulin fusion polypeptide. This sequence was verified by D ⁇ A dideoxy sequencing.
  • the peptide fused to ⁇ 2microglobulin in the protein expressed from pEX013, AAKAKAAAL, consists of Alanine residues in all positions except positions 3, 5 and 9 which are optimised for anchoring the peptide to the HLA-B*0801 heavy chain.
  • Example 4 DNA plasmid vector for expression of an HLA-A2 single position randomised peptide-- ⁇ 2microglobulin fusion in E.coli.
  • Examples 4-11 describe the design and methods used for generating D ⁇ A plasmids from which randomised peptide- ⁇ 2microglobulin fusions, suitable for forming complexes with HLA-A2 and HLA-B8 heavy chains, can be expressed. The same principles can be applied to peptide sequences suitable for loading into any class I or class JJ HLA heavy chain molecule.
  • Example 4 describes the constmction of vectors for the expression of peptide- ⁇ 2microglobulin fusions, in which the peptide part contains HLA-A2 anchor residues and in which one position in the peptide could be occupied by any one of sixteen different amino acids.
  • Example 5 describes a similar approach whereby fusions in which two positions in the peptide are randomised, producing a total peptide variation of 256.
  • Example 6 describes the construction of vectors to produce fusions in which three positions in the peptide are randomised, producing a total peptide variation of 4096.
  • Example 7 describes the constmction of vectors to produce fusions in which seven positions in the peptide are randomised, producing a total peptide variation of 2.7 x ⁇ 10 8 .
  • Examples 8-11 describe the equivalent strategies for HLA-B8.
  • the approaches described can be easily adapted to produce fusion proteins in which further residues in the peptide part are randomised. It can also easily be adapted to produce fusion proteins in which positions other than those chosen in Examples 4-11 are randomised. However, the preferred options are to randomise one, two or three positions in the peptide. The reason for this is that, although a higher degree of variation is likely to include antigen complexes that stimulate a larger proportion of the T cells, a certain concentration of each particular antigen may also be required. Thus, an increase in the degree of variation in antigens has to be balanced against a loss of concentration of the individual antigen.
  • alloreactive T cells are likely to depend significantly less on peptide identity than is the case for conventional T cell responses, and so even a limited number of antigen complexes may be able to stimulate a significant proportion of the alloreactive T cells present in a sample.
  • a PCR reaction is performed with vector pEX013 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3').
  • the forward primer contains redundant bases in three positions and has the following sequence:
  • the peptide-linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture (plasmid library) have a redundancy of factor of 16. All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • HLA-A2 anchor residues at positions 2 and 9, are underlined.
  • "Ran” is the position which is randomised and corresponds to position 5 in HLA-A2 peptide ligand. Due to the redundancy in the PCR primer, position 5 ("Ran") in the peptide could be occupied by any of the following amino acids: Leucine, Proline, Histidine, Glutamine, Arginine, Isoleucine, Methionine, Threonine, Asparagine, Lysine, Serine, Naline, Alanine, Aspartic Acid, Glutamic Acid, Glycine.
  • Resides that are excluded from being present at position 5 are: Phenylalanine,
  • Example 5 DNA plasmid vector for expression of an HLA-A2 double position randomised peptide- ⁇ 2microglobulin fusion in E.coli.
  • a PCR reaction is performed with vector pEX013 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3').
  • the forward primer contains redundant bases in six positions and has the following sequence: 5' GGGGGG CATATGGCACGT GCT GCGXNNXNNGCAGCG GTT GGA TCC GGT GGG 3',
  • the resulting vectors have a sequence redundancy factor of 2304.
  • the peptide-linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture (plasmid library) have a redundancy of factor of 256. All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • HLA-A2 anchor residues at positions 2 and 9, are underlined.
  • “Ran” indicates the positions which are randomised (positions 5 and 6 in HLA-A2 peptide ligand). Due to the redundancy in the-PCR primer, positions 5 and 6 in the peptide may be occupied by any of sixteen different amino acids as described in Example 5.
  • Example 6 DNA plasmid vector for expression of an HLA-A2 triple position randomised peptide- ⁇ 2microglobulin fusion in E.coli.
  • PCR reaction is performed with vector pEX013 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3 ').
  • the forward primer contains redundant bases in nine positions and has the following sequence:
  • the resulting vectors have a sequence redundancy factor of 110592.
  • the peptide- linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture (plasmid library) have a redundancy of factor of 4096. All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • Example 7 A DNA plasmid vector for expression of an HLA-A2 seven position randomised peptide— ⁇ microglobulin fusion in E.coli.
  • PCR reaction is performed with vector pEX013 (see Example 2) as template and the same reverse primer as used in Example 1 (5'- GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG -3 ').
  • the forward primer contain redundant bases in nine positions and has the following sequence:
  • the resulting vectors have a sequence redundancy factor of -5.8x10 .
  • the peptide- linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture have a sequence redundancy factor of -5.8x10 .
  • Plasmid library have a redundancy of factor of ⁇ 2.7xl0 . All variation is m the peptide part of the fusion polypeptide which has the following sequence:
  • HLA-A2 anchor residues at positions 2 and 9, are underlined.
  • 'Ran' indicates the positions which are randomised and corresponds to positions 1, 3, 4, 5, 6, 7 and 8 in HLA-A2 peptide ligands. Due to the redundancy in the PCR primer, these positions in the peptide could each be occupied by sixteen different amino acids as described in Example 4.
  • Example 8 A DNA plasmid vector for expression of an HLA-B8 single position randomised peptide- ⁇ 2microglobulin fusion in E.coli.
  • a PCR reaction is performed with vector pEX014 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3').
  • the forward primer contains redundant bases in three positions and has the following sequence:
  • the peptide-linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture (plasmid library) have a redundancy of factor of 16. All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • Example 9 A DNA plasmid vector for expression of an HLA-B8 double position randomised peptide- ⁇ 2microglobulin fusion in E.coli.
  • PCR reaction is performed with vector pEX014 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3').
  • the forward primer contains redundant bases in six positions and has the following sequence:
  • the resulting peptide-linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture have a redundancy of factor of 256. All variation is in the peptide part of the fusion polypeptide which has the following sequence: (Met) -Ran - Ala - Lys - Ala- Lys - Ala- Ran - Ala - Leu
  • HLA-B8 anchor residues at positions 3, 5 and 9, are underlined.
  • “Ran” indicates the positions which are randomised (positions 1 and 7 in HLA-B8 peptide ligand). Due to the redundancy in the PCR primer, position 1 and 7 in the peptide are each occupied by any one of sixteen different amino acids as described in Example 4.
  • Example 10 A DNA plasmid vector for expression of an HLA-B8 triple position randomised eptide- ⁇ 2microglobulin fusion in E.coli.
  • PCR reaction is performed with vector pEX014 (see Example 2) as template and the same reverse primer as used in Example 1 (5' GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG 3 ').
  • the forward primer contains redundant bases in nine positions and has the following sequence:
  • the resulting vectors have a sequence redundancy factor of 110592.
  • the peptide- linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture have a sequence redundancy factor of 110592.
  • Plasmid library have a redundancy of factor of 4096. All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • Example 11 A DNA plasmid vector for expression of an HLA-B8 six position randomised peptide— ⁇ 2microglobulin fusion in E.coli.
  • PCR reaction is performed with vector pEX014 (see Example 3) as template and the same reverse primer as used in Example 1 (5'- GGG GGG GAA TTC AAG CTT ACA TGT CTC GAT CCC ACT TAA CTA TCT TG -3').
  • the forward primer contains redundant bases in eighteen positions and has the following sequence:
  • the resulting vectors have a sequence redundancy factor of ⁇ 1.2xl0 10 .
  • the peptide- linker- ⁇ 2microglobulin fusion polypeptides expressed from the plasmid mixture (plasmid library) have a redundancy of factor of ⁇ 1.7xl0 7 . All variation is in the peptide part of the fusion polypeptide which has the following sequence:
  • Example 12 Synthetic peptides randomised at one, two or three positions and suitable as HLA-A2 antigens for recognition by alloreactive T cells
  • Examples 12 and 13 describe designs for synthesis of mixed peptides which can used for detecting T cell alloreactivity against HLA-A2 and HLA-B8 respectively.
  • Soluble HLA complexes can be produced with synthetic peptide ligands. Peptide synthesis allows the incorporation of any selection of amino acids at individual positions. Thus, highly selected peptide mixes are used for generating HLA complexes suitable for the detection of alloreactive T cell activity. Synthetic peptides can be made to order by a number of companies, for example, Research Genetics Inc., Huntsville, AL USA.
  • a mixed peptide antigen, randomised at a single position, and for presentation by HLA-A2 has the following sequence:
  • a mixed peptide antigen, randomised at two positions, and for presentation by HLA- A2 has the following sequence:
  • a mixed peptide antigen, randomised at three positions, and for presentation by HLA- A2 has the following sequence:
  • a mixed peptide antigen, randomised at seven positions, and for presentation by HLA- A2 has the following sequence:
  • Example 13 Synthetic peptides randomised at one, two or three positions and suitable as HLA-B8 antigens for recognition by alloreactive T cells
  • Synthetic peptides with randomised positions are synthesised for other HLA molecules as described in Example 12.
  • HLA-B8 a mixed peptide antigen, randomised at a single position, has the following sequence:
  • a mixed peptide antigen, randomised at two positions, and for presentation by HLA- B8 has the following sequence:
  • a mixed peptide antigen, randomised at three positions, and for presentation by HLA- B8 has the following sequence:
  • a mixed peptide antigen, randomised at six positions , and for presentation by HLA- B8 has the following sequence:
  • Peptide - ⁇ 2m fusions were expressed from the DNA plasmid pBJ196 and mutated derivatives thereof such as pEX013 described in Examples 1-11. These encode fusion proteins comprising the 'flu matrix peptide (or mutated versions thereof), a flexible linker sequence mainly comprised of Glycine and Serine, and - ⁇ 2m, in a transformation competent strain of E. coli (a number of which are commercially available from Novagen, Madison, WI, USA).
  • the plasmid pBJ196 contains the ⁇ eptide- ⁇ 2m fusion under the control of the strongly inducible T7 promoter in the vector pGMT7 (Studier, et al.
  • E. coli BL21 cells transformed with one of the peptide - ⁇ 2m fusion vectors were plated on LB/agar/100 mg/ml ampicillin plates made according to a standard recipe. Transformants were then grown in TYP medium with Ampicillin (16 g/1 Bacto- Tryptone, 16 g/1 Yeast Extract, 5 g/1 NaCl, 2.5 g/1 K2HPO4, 100 mg/1 Ampicillin) to an OD600 ⁇ 0.4.
  • TYP medium with Ampicillin (16 g/1 Bacto- Tryptone, 16 g/1 Yeast Extract, 5 g/1 NaCl, 2.5 g/1 K2HPO4, 100 mg/1 Ampicillin) to an OD600 ⁇ 0.4.
  • 1 1 volumes of TYP media were prepared in 2 1 conical flasks and these were covered with four layers of aluminium foil prior to being autoclaved. Cell densities were measured using optical density at 600 nm wavelength (OD600) on a Beckman DU530 spectrop
  • Inclusion bodies were purified as described (Gao, et al, Prot. Sci.7: 1245-49 (1998)). Cells were lysed in 'Lysis Buffer' (10 mM EDTA (from 0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodium acetate pH 5.2, stored at -20°C), 10 mM Tris pH 8.1 (from 2 M stock pH 8.1), 150 mM NaCl (from 4 M stock), 200 mg/ml lysozyme (from 20 mg/ml stock stored at -20°C), 10% glycerol (from fluid), 2500 units of DNAase I and lOmM MgCl 2 using a 50 ml Dounce homogeniser, (DNase I and lysozyme were from Sigma).
  • 'Lysis Buffer' 10 mM EDTA (from 0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 m
  • the inclusion bodies were separated from cell debris by centrifugation in a Beckman J2-21 centrifuge equipped with a JA-20 rotor as described (Gao, et al, Prot. Sci.7: 1245-49 (1998)) and stored at -20°C.
  • Inclusion bodies were then thawed and resuspended in 'Resuspension Buffer' (50 mM Tris pH 8.1 (from 2 M stock), 100 mM NaCl (from 4 M stock), 10 mM EDTA (from 0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodium acetate pH 5.2, stored at -20°C)), and denatured in 'Denaturing Buffer' (6M Guanidine and lOmM DTT buffered with Tris-HCl pH 8.1 all chemicals from Sigma).
  • 'Resuspension Buffer' 50 mM Tris pH 8.1 (from 2 M stock), 100 mM NaCl (from 4 M stock), 10 mM EDTA (from 0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodium acetate pH 5.2, stored at -20°C)
  • 'Denaturing Buffer' 6M Guanidine
  • HLA complexes were folded using 30 mg of heavy chain protein and 25 mg of ⁇ 2 microglobulin-peptide fusion protein.
  • the MHC complexes were biotinylated using 5 ⁇ g/ml purified BirA enzymes, 0.5 mM biotin and 5 mM ATP. The reaction was incubated at room temperature for 16 h. MHC-peptide complexes were recovered by FPLC purification and ion exchange chromatography. Tetramers were made by mixing biotinylated complexes with streptavidin-P ⁇ (Sigma Chemicals Co) at a molar ratio of 4: 1. The labelled tetramers were concentrated to 3-4mg/ml and stored in PBS at 4°C.
  • HLA complexes were folded using 30 mg of heavy chain protein, 25 mg of ⁇ 2 microglobulin and 10 mg of peptide.
  • the MHC complexes were biotinylated using 5 ⁇ g/ml purified BirA enzymes, 0.5 mM biotin and 5 mM ATP. The reaction was incubated at room temperature for 16 h. MHC-peptide complexes were recovered by FPLC purification and ion exchange chromatography. Tetramers were made by mixing biotinylated complexes with streptavidin-P ⁇ (Sigma Chemicals Co) at a molar ratio of 4:1. The labelled tetramers were concentrated to 3-4mg/ml and stored in PBS at 4°C.
  • T cell activation is detected using the following reagent and methods.
  • ELISPOT kit for human IFN- ⁇ from MABTECH (cat.no. 3420-2 ALP). Lymphoprep from Nycomed Pharma AS (cat. no. 1031966). Microfilter plates from Milipore (cat. no. MAIP s45 10). Alkaline phosphatase conjugate substrate from BIORAD (cat. no.
  • Assays are performed in duplicate with appropriate positive and negative controls.
  • a peptide concentration of 2 ⁇ M is used for octamers/nonamers with incubation o/n at 37 °C and a flick of cells and washing x6 with PBS plus 0.05% Tween.
  • 50 ⁇ l anti-IFN- ⁇ -biotin diluted in PBS is added and incubation at room temperature for 3 hr is carried out followed by washing x6 with PBS plus 0.05% Tween.
  • 50 ⁇ l streptavidin conjugated alkaline phosphatase (AP) diluted in PBS is added and incubation at room temperature for 1 hr followed by washing x6 with PBS plus 0.05% Tween.
  • 100 ⁇ l of colour reagent is added followed by incubation for 20-30 minutes and washing xl with tap water. Water is flicked out and the plate is dried. Spots are counted using a low power microscope with an eyepiece grid.

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Abstract

La présente invention concerne une méthode permettant de déterminer si un lymphocyte T réagit à un type déterminé de complexe majeur d'histocompatibilité (CMH). Dans le cadre de cette méthode, on met un lymphocyte T en contact avec une pluralité de molécules dudit type CMH, chaque molécule CMH étant complexée avec un antigène peptidique dont le rôle dans la liaison du récepteur de lymphocyte T avec le complexe antigène peptidique est réduit au minimum. On détermine ensuite si ladite pluralité de molécules CMH provoque une activation du lymphocyte T. Cette méthode permet d'évaluer l'activité allo-spécifique de lymphocytes T et peut être utilisée pour apparier des donneurs et des receveurs de greffons ainsi que pour surveiller des réponses allo-réactives à la suite d'une transplantation.
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US8715680B2 (en) 2006-05-09 2014-05-06 The University Of Birmingham HLA peptide therapy
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
WO2008139163A1 (fr) * 2007-05-09 2008-11-20 Circassia Limited Analyse pronostique pour déterminer une réponse de lymphocyte t à des antigènes hla et utilisation de celle-ci dans le domaine de la transplantation tissulaire
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US11402373B2 (en) 2014-06-13 2022-08-02 Immudex Aps General detection and isolation of specific cells by binding of labeled molecules
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US12258373B2 (en) 2018-12-17 2025-03-25 Immudex Aps Panel comprising Borrelia MHC multimers
WO2021087579A1 (fr) * 2019-11-08 2021-05-14 The University Of Sydney Compositions pour la détection de lymphocytes t alloréactifs

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